Induction of protective immunity against antigens

ABSTRACT

Described herein are compositions and methods for making and using recombinant bacteria that are capable of regulated attenuation and/or regulated expression of one or more antigens of interest.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/449,228, filed on Jan. 23, 2017 and U.S. Provisional Application No.62/541,293, filed on Aug. 4, 2017. The entire content of each of theforegoing applications is expressly incorporated by reference herein.

This invention was made with government support under Grant Nos.AI093348, AI056289 and AI126712, awarded by The National Institutes ofHealth. The government has certain rights in the invention.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jan. 23, 2018 and is 31 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND

Salmonella enterica causes heavily burdened diseases in humansworldwide. S. Typhi and Paratyphi A, B and C cause enteric fever (1) andare major public health concerns (2-4). S. Typhi is estimated to causeover 20.6 million cases, 433,000 deaths globally each year (5, 6) and12.2 million disability-adjusted life years (7). In addition to theseserovars, nontyphoidal Salmonella (NTS) is increasingly being recognizedas important causes of invasive diseases (2, 8, 9), such as sepsis andmeningitis, with 93.8 million cases and 681,300 deaths annually globally(10, 11). NTS is also a leading cause of hospitalization and death fromfood borne disease in the US (12), ˜1.2 million cases of inflammatorydiarrheal disease per year, resulting in 23,000 hospitalizations and 450deaths (12, 13) with an economic loss of approximately $3.31 billion dueto premature mortality, disability, medical and productivity costs andan annual loss of 16,782 quality-adjusted life years (14). Amongchildren <5 years old, NTS is the top bacterial pathogen and causes 4670hospitalization and 38 deaths (15). NTS disease in the US is accountedprimarily by serovars belonging to three serovars B, D and C (16).Serovars Enteritidis, Typhimurium, Newport, and Heidelberg are the mostcommon outbreaks in the US (17). Though the vast majority of patientsdevelop self-limiting gastroenteritis, characterized by inflammatorydiarrhea, NTS can also cause systemic diseases and is the single mostcommon cause of death from food-borne illnesses associated with viruses,parasites or bacteria in the US primarily in immunocompromised persons(18). In young children and HIV-infected individuals, NTS frequentlycauses systemic infection that is associated with high mortality (19).The rise of AIDS in many parts of the world, notably in sub-SaharanAfrica, has resulted in a dramatic increase in the frequency ofNTS-associated systemic infection (20, 21). Bacteremia is the mostsevere symptom and mortality in bacteremic children who reach a cliniccan be nearly 25% (18, 21). Enteric fever and NTS become increasinglydifficult to treat with antibiotics because of the rise in Salmonella ofmulti-drug resistance (22, 23), leading to the risk of an increasingnumber of untreatable cases (24, 25).

Enteric fever can be prevented with several vaccines (26, 27). Killedwhole cell preparations of serovars Typhi and Paratyphi were effectivein diminishing the incidence in endemic areas (28), but werediscontinued due to frequent adverse reactions (29). A live attenuatedS. Typhi strain Ty21a, generated by chemical mutagenesis, confers only amoderate level of protection for up to three years against serovarTyphi, but not other relevant serovars (29, 30). Additional geneticallymodified Salmonella strains have been tested in clinical trials withsome success, but none of them has been approved. The purified capsularcarbohydrate Vi of serovar Typhi induces protective immunity overseveral years against Typhi and possibly Paratyphi C, but not againstParatyphi A and B or Typhimurium that all lack this capsule (31).Conjugation of Vi with a protein antigen improves immune responses ininfants, a major susceptible population for enteric fever.

To cover the important serovar Paratyphi A, current efforts focus onlinking the O-antigen, the carbohydrate part of lipopolysaccharide(LPS), with a protein antigen (27). These two commercial vaccines aremainly used for the traveler vaccine market and no new vaccine forwidespread use has been licensed since the 1990s (26). Although threetypes of vaccines against S. Typhi are currently commercially available,unfortunately, there is still not a single licensed vaccine availableagainst S. Paratyphi A, with very little, if any cross-protectionprovided by the available S. Typhi vaccines. There are vaccines againstNTS serovars Enteritidis and Typhimurium which are effective in farmanimals, like poultry and pigs (32), but not available in humans (33).This represents a significant limitation in the existing preventionstrategies. Therefore, treatment for systemic salmonellosis has becomeincreasingly difficult, and current vaccines against Salmonella onlyprovide at moderated levels, limited duration of protection, and limitedcoverage of clinically relevant serovars. These situations generate anurgent medical need for improved Salmonella vaccines.

The use of recombinant attenuated Salmonella vaccines (RASVs) as avaccine or a heterologous antigen delivery system has been studiedbecause of their abilities to stimulate systemic and mucosal immuneresponses at local and distal sites and advantages as vectors to produceand present recombinant vaccine antigens. RASVs can be used for amultitude of applications including, but not limited to, vaccinationagainst pathogens that cause disease, cancer, chronic respiratorydisease, and heart disease. Recently, Regulated Delayed Attenuated RASVs(RDA RASVs) have been developed to enhance the immune responses to RASVsand the protective antigen carried. RDA RASVs are engineered so thatgenes for key virulence factors are under the control of an induciblepromoter P_(araBAD), induced by arabinose not found in the mammalianhost. The RDA RASVs are grown in vitro in the presence of arabinose sothat genes mediating the pathogenic phenotype are expressed and RASVsdisplay features of wild-type to invade into the hosts. Expression ofthe pathogenic genes ceases due to the absence of arabinose in vivo,with gene products diluted due to replication, producing an attenuatedphenotype without causing disease. Since they replicate initially withfull virulence, they colonize lymphoid tissues to higher levels toelicit more potent immune responses than a constitutively attenuatedRASV.

Regulated delayed protein synthesis (RDPS) have also been developed toenhance immunogenicity. The increased antigen synthesis levels help toincrease the chance that cognate T cells interact with antigenpresentation cells (APCs), leading to effective proliferation andproduction of effector molecules and T-cell proliferation in vivo.However, high-level antigen synthesis imposes metabolic demands thatimpair the strains' ability to colonize effector lymphoid tissues. AnRDPS system makes recombinant vaccine antigen production only after theRASV colonizes lymphoid tissues as the RASV cells multiply in vivo. Thisstrategy is not influenced by the mode of attenuation.

Although the use of recombinant Salmonella as live vaccines to producean immune response in subjects is promising, the organisms are live andsometimes pathogenic. Accordingly, it is necessary to introduceregulatory systems into the bacteria to attenuate and control theexpression of antigens that are expressed by the bacteria. The currentlyutilized means of attenuation make live vaccine strains susceptible toenvironmental stresses in vivo. Consequently, fewer bacteria are able tocolonize the host cell in order to achieve a desirable level ofimmunogenicity. Thus, there is a need for new strains of recombinantmicroorganisms that can be developed for use as live vaccines, which areless susceptible to environmental stresses in vivo and which cancolonize host cells in order to achieve better levels of immunogenicity.There is also a need for new means to enhance the safety of liveattenuated vaccines in vivo.

SUMMARY

The instant disclosure provides strains of recombinant bacteria,including Salmonella, which depend on three sugars to regulate thevirulence phenotype of the bacteria by controlling the expression ofmultiple virulence genes and of an antigen of interest, as well as aregulated delayed lysis phenotype, allowing for biological containmentand the enhancement of immunogenic properties. Other attributes that canbe regulated by one or more of the sugars includes acid tolerance during(e.g., during oral immunization) as described in U.S. Patent ApplicationPublication No. 2014/0370057, the entire contents of which are expresslyincorporated herein by reference. The dependence on three sugarsenhances the safety of the recombinant bacteria, given the improbabilitythat the organisms will encounter all three sugars in anaturally-occurring environment. Surprisingly, the instant inventiondemonstrates that three distinct sugars could successfully be used toregulate attributes of the recombinant bacteria (e.g., the expression ofgenes encoding an antigen of interest, delayed lysis phenotype and/orvirulence gene expression) without cross-interference of any one sugarin the sugar-regulatable activity of any other sugar by cataboliterepression.

The organisms can be used for the safe and highly effective delivery ofantigenic compounds to a subject in order to mount effective protectiveimmune responses. Such recombinant bacteria can manipulate cell surfacesynthesizing protective antigens and can induce protective immuneresponses to multiple Salmonella serovars. The recombinant bacteria canbe used to enhance survival of the bacteria to host defense stressessuch as stomach acid; to confer regulated delayed attenuation; to conferregulated-delayed lysis in vivo (e.g., by control of asdA and murA geneexpression with release of an antigen of interest or of a DNA vaccineencoding them); or to enable fusion of carbohydrate polymers ontocarbohydrate and/or proteins.

Specifically, disclosed herein are triple sugar regulated RecombinantAttenuated Salmonella Vaccine (RASV) strains. These strains delivermultiple conserved protective Salmonella surface/secreted antigens withtheir natural conformations to induce protective immunity againstmultiple virulent Salmonella serovars. As an example, the RASVs may havea rhamnose-regulated O-antigen synthesis, combined with amannose-regulated O-antigen side chain synthesis to expose conservedinner core, and an arabinose-regulated production of Generalized Modulesfor Membrane Antigens (GMMA), or outer membrane vesicles, in vivo forenhancing production of conserved outer membrane proteins (OMPs). RASVsmay be constructed in two Salmonella serovars, group B S. Typhimuriumand group D S. Enteritidis, to express conserved immunogen genes and tomaximize anti-Salmonella humoral, cellular and mucosal immune responses.The disclosed RASVs have rational design features different from otherRASVs that enhance success. Specifically, the disclosed RASVs providesafe and highly effective Salmonella vaccines with low cost and can beused to develop S. Typhi or S. Paratyphi A RASVs for human use.

In one aspect, the disclosure provides a recombinant derivative of apathogenic bacterium comprising: a.) a first gene regulated by a firstsugar which confers a first phenotype; b.) a second gene regulated by asecond sugar which confers a second phenotype; and c.) a third generegulated by a third sugar which confers a third phenotype; wherein thefirst, second and third phenotypes are selected from the groupconsisting of: 1. a regulated-delayed attenuation; 2. aregulated-delayed expression of an antigen of interest; 3. aregulated-delayed lysis in vivo; 4. a regulated synthesis of O-antigen;5. a regulated synthesis of an O-antigen side chain; 6. a regulatedproduction of Generalized Modules for Membrane Antigens (GMMA); 7.regulated enhanced survival to a host stress condition; and 8. aregulated production of outer membrane vesicles (OMVs).

In one aspect, the disclosure provides a recombinant derivative of apathogenic bacterium comprising: a.) a first gene regulated by a firstsugar which confers a first phenotype; b.) a second gene regulated by asecond sugar which confers a second phenotype; and c.) a third generegulated by a third sugar which confers a third phenotype; wherein thefirst, second and third phenotypes are selected from the groupconsisting of: 1. a regulated-delayed attenuation; 2. aregulated-delayed expression of an antigen of interest; 3. aregulated-delayed lysis in vivo; 4. a regulated synthesis of O-antigen;5. a regulated production of Generalized Modules for Membrane Antigens(GMMA); 6. regulated enhanced survival to a host stress condition; and7. a regulated production of outer membrane vesicles (OMVs).

In one embodiment, the first sugar, second sugar, and third sugar areeach a different sugar. In one embodiment, the first sugar, secondsugar, or third sugar does not interfere with the regulation of a generegulated by a different sugar.

In one embodiment, the first sugar is selected from the group consistingof arabinose, mannose, xylose, galactose, rhamnose, and maltose. In oneembodiment, the second sugar is selected from the group consisting ofarabinose, mannose, xylose, galactose, rhamnose, and maltose. In oneembodiment, the third sugar is selected from the group consisting ofarabinose, mannose, xylose, galactose, rhamnose, and maltose.

In one embodiment, the first gene is operably-linked to a firstsugar-regulatable promoter. In one embodiment, the second gene isoperably-linked to a second sugar-regulatable promoter. In oneembodiment, the third gene is operably-linked to a thirdsugar-regulatable promoter.

In one embodiment, a gene is modified to enable a reversible synthesisof a sugar-containing molecule that confers a sugar regulatablephenotype. In one embodiment, the modified gene ispmi. In oneembodiment, the modified gene is galE.

In one embodiment, the bacterium is a Gram-negative bacterium. In oneembodiment, the bacterium belongs to the family Enterobacteriaceae.

In one embodiment, the phenotype is regulated-delayed attenuation, andthe gene conferring the phenotype is fur. In one embodiment, thephenotype is regulated-delayed expression of an antigen of interest, andthe gene conferring the phenotype encodes an antigen of interest. In oneembodiment, the phenotype is the regulated-delayed lysis in vivo,wherein the lysis is enabled to occur in a cytosol due to mutation in asifA gene. In one embodiment, the phenotype is regulated synthesis ofO-antigen, and the gene conferring the phenotype is selected from thegroup consisting of waaG, rfaH, waaJ, wbaP, wzy, waaP, waaO, waaF, waaP,waaC, waaA, waaL and wbaP. In one embodiment, the phenotype isproduction of Generalized Modules for Membrane Antigens (GMMA) or outermembrane vesicles, and the gene conferring the phenotype is selectedfrom the group consisting of ybgC, tolQ, tolA, tolR, tolB, paI, andybgF.

In one embodiment, the phenotype is regulated synthesis of O-antigenside chain, and the gene conferring the phenotype is tolR. In oneembodiment, the first phenotype is regulated O-antigen synthesis and thesecond phenotype is production of GMMA or outer membrane vesicles.

In one embodiment, the bacterium further comprises a gene encoding anantigen of interest not operably-linked to a sugar regulatable promoter.

In one embodiment, the bacterium comprises a deletion of an endogenousO-antigen synthesis gene. In one embodiment, the deletion in theendogenous O-antigen synthesis gene comprises a partial deletion of thegene. In one embodiment, the deletion in the endogenous O-antigensynthesis gene comprises a full-length deletion of the gene. In oneembodiment, the O-antigen synthesis gene is waaL or wbaP.

In one embodiment, the bacterium comprises a deletion in an endogenousphosphomannose isomerase gene. In one embodiment, the deletion in theendogenous phosphomannose isomerase gene comprises a partial deletion ofthe gene. In one embodiment, the deletion in the endogenousphosphomannose isomerase gene comprises a full-length deletion of thegene. In one embodiment, the phosphomannose isomerase gene is pmi.

In one embodiment, the bacterium comprises a deletion in an endogenoustol-paI system gene. In one embodiment, the deletion in the endogenoustol-paI system gene comprises a partial deletion of the gene. In oneembodiment, the deletion in the endogenous tol-paI system gene comprisesa full-length deletion of the gene. In one embodiment, the endogenoustol-paI system gene is selected from the group consisting of ybgC, tolQ,tolA, tolR, tolB, paI, and ybgF.

In one embodiment, the first gene, second gene and/or third gene islocated on a plasmid in the bacterium. In one embodiment, the firstgene, second gene and/or third gene is located on a chromosome in thebacterium.

In one embodiment, the first, second or third sugar-regulatable promoteris a rhamnose-regulatable promoter. In one embodiment, therhamnose-regulatable promoter is rhaSR P_(rhaBAD).

In one embodiment, the first, second or third sugar-regulatable promoteris an arabinose-regulatable promoter. In one embodiment, the arabinoseregulatable promoter is araC P_(araBAD).

In one embodiment, the bacterium further comprises a deletion in anendogenous relA gene. In one embodiment, the deletion of the endogenousrelA gene is a partial deletion of the gene. In one embodiment, thedeletion of the endogenous relA gene is a full-length deletion of thegene.

In one embodiment, the bacterium further comprises a nucleic acidencoding a LacI repressor. In one embodiment, the LacI repressor isencoded by a lacI gene. In one embodiment, the nucleic acid encoding theLacI repressor is located on a plasmid in the bacterium. In oneembodiment, the nucleic acid encoding the LacI repressor is located on achromosome in the bacterium.

In one embodiment, the bacterium further comprises a deletion in anendogenous P_(fur) promoter.

In one embodiment, the fur gene is operably-linked to anarabinose-regulatable promoter. In one embodiment, the fur gene islocated on a plasmid in the bacterium. In one embodiment, the fur geneis located on a chromosome in the bacterium.

In one embodiment, the bacterium further comprises a deletion in geneencoding an aspartate-semialdehyde dehydrogenase. In one embodiment, thegene encoding the aspartate-semialdehyde dehydrogenase comprises an asdgene. In one embodiment, the gene encoding the aspartate-semialdehydedehydrogenase comprises an asdA gene.

In one embodiment, the gene encoding the antigen of interest is locatedin a plasmid in the bacterium. In one embodiment, the plasmid furthercomprises a nucleic acid encoding an aspartate-semialdehydedehydrogenase. In one embodiment, the aspartate-semialdehydedehydrogenase comprises AsdA. In one embodiment, the plasmid is a lowcopy number plasmid. In one embodiment, the plasmid is a high copynumber plasmid. In one embodiment, the plasmid is selected from thegroup consisting of pYA4589, pYA4595, pYA4763, pG8R15, pG8R16, pG8R17,pG8R18, pGR111, pG8R112, pG8R113, and pG8R114.

In one embodiment, the gene encoding the antigen of interest is locatedon a chromosome in the bacterium.

In one embodiment, the bacterium further comprises a deletion in apagLgene. In one embodiment, the deletion of the pagL gene is a partialdeletion of the gene. In one embodiment, the deletion of the pagL geneis a full-length deletion of the gene. In one embodiment, the mutationis ΔwaaL/ΔpagL::TT rhaSR P_(rhaBAD) waaL.

In one embodiment, the bacterium further comprises an antigen ofinterest operably-linked to a repressor-regulatable promoter. In oneembodiment, the promoter is a lactose-regulatable promoter. In oneembodiment, the lactose-regulatable promoter is a LacI-regulatablepromoter. In one embodiment, the LacI-regulatable promoter is selectedfrom the group consisting of P_(trc), P_(lac), P_(T7lac), P_(tac),P_(ompA lacO), and P_(lpp lacO).

In one embodiment, the antigen of interest is an antigen derived from aninfectious agent. In one embodiment, the antigen of interest is derivedfrom an infectious agent selected from the group consisting of a virus,a bacterium, a protozoan, a prion, a fungus, and a helminth. In oneembodiment, the antigen of interest is derived from a bacterium. In oneembodiment, the antigen of interest is a Salmonella antigen. In oneembodiment, the Salmonella antigen is selected from the group FliC,FliC180, OmpC, OmpD, OmpF, SseB, and SseI. In one embodiment, theantigen of interest is an antigen from a Clostridium bacterium. In oneembodiment, the antigen is a C. perfringens antigen. In one embodiment,the antigen comprises NetB, PlcC, antigenic fragments thereof, fusionproteins comprising said antigens, or fusion proteins comprisingantigenic fragments of antigens.

In one embodiment, the antigen of interest is a viral antigen. In oneembodiment, the antigen of interest is an influenza antigen. In oneembodiment, the influenza antigen is hemagglutinin or neuraminidase.

In one embodiment, the antigen of interest is an antigen associated withcancer. In one embodiment, the antigen associated with cancer isselected from the group consisting of MAGE-A, MAGE-C1, BAGE, GAGE, CAGE,XAGE, NY-ESO1, LAGE1, and survivin.

In one embodiment, the antigen is a protein antigen encoded by a nucleicacid sequence codon optimized for expression in said bacterium.

In one embodiment, the bacterium further comprises a deletion in a sifAgene. In one embodiment, the deletion of the sifA gene is a partialdeletion of the gene. In one embodiment, the deletion of the sifA geneis a full-length deletion of the gene. In one embodiment, the sifA geneis operably-linked to an arabinose-regulatable promoter.

In one embodiment, the bacterium further comprises a deletion in a recFgene. In one embodiment, the deletion of the recF gene is a partialdeletion of the gene. In one embodiment, the deletion of the recF geneis a full-length deletion of the gene.

In one embodiment, the bacterium further comprises a deletion in a recJgene. In one embodiment, the deletion of the recJ gene is a partialdeletion of the gene. In one embodiment, the deletion of the recJ geneis a full-length deletion of the gene.

In one embodiment, the bacterium is of the genus Salmonella. In oneembodiment, the bacterium is a Salmonella enterica bacterium. In oneembodiment, the bacterium is a Salmonella enterica subsp. entericaserovar Paratyphi A bacterium, a Salmonella enterica subsp. entericaserovar Enteritidis bacterium, a Salmonella enterica subsp. entericaserovar Typhi bacterium, a Salmonella enterica subsp. enterica serovarTyphimurium bacterium, Salmonella enterica subsp. enterica serovarDublin, or Salmonella enterica subsp. enterica serovar Choleraesuis.

In another aspect, disclosed herein is a pharmaceutical compositioncomprising a recombinant bacterium disclosed herein, and apharmaceutically acceptable carrier.

In another aspect, disclosed herein is a method for eliciting an immuneresponse against an antigen of interest in a subject, the methodcomprising administering to the subject an effective amount of apharmaceutical composition disclosed herein.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Depicts three vectors containing the sugar-regulated cassettesaraC P_(araBAD), rhaRS-P_(raBAD) and xylR-P_(xylA) to enableconstruction of a suicide vector derivative to generate fusions of asugar regulation cassette to a gene of interest for the replacement ofthe native promoter for that gene of interest.

FIG. 2 depicts three plasmids in which GFP synthesis is regulated bythree different sugars.

FIGS. 3A, 3B, and 3C depict galactose-insensitive mutationA(galE-ybhC)-851.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H depict growth curves ofSalmonella strains with different galE mutations in Nutrient broth withvarying concentrations of galactose.

FIG. 5 depicts the colonization of galE mutants.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, 6J, 6K, 6L, 6M, 6N, 6O, 6P,6Q, and 6R depict growth curves of Salmonella strains χ12341(pYA4763)and χ3761 during 24 h in growth media with varying sugar concentrationsas indicated.

DETAILED DESCRIPTION

In order that the disclosure may be more readily understood, certainterms are first defined. These definitions should be read in light ofthe remainder of the disclosure and as understood by a person ofordinary skill in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by a person of ordinary skill in the art. Additionaldefinitions are set forth throughout the detailed description.

As used herein, the term “recombinant bacterium” refers to a bacterialcell that has been genetically modified from its native state. Forinstance, a recombinant bacterium may comprise one or more nucleotideinsertions, nucleotide deletions, nucleotide rearrangements, andnucleotide modifications. These genetic modifications may be introducedinto the chromosome of the bacterium, or alternatively be present on anextrachromosomal nucleic acid (e.g., a plasmid). Recombinant bacteria ofthe disclosure may comprise a nucleic acid located on a plasmid.Alternatively, the recombinant bacteria may comprise a nucleic acidlocated in the bacterial chromosome (e.g., stably incorporated therein).In some embodiments, the recombinant bacterium is avirulent. In someembodiments the recombinant bacterium exhibits reduced virulence. Insome embodiments, the recombinant bacterium is non-virulent. In someembodiments, the recombinant bacterium is pathogenic. In someembodiments, the recombinant bacterium is attenuated. In anotherembodiment, the recombinant bacterium is a recombinant derivative of apathogenic bacterium.

As used herein, the term “gene” refers to a nucleic acid fragment thatencodes a protein or a fragment thereof, or a functional or structuralRNA molecule, and may optionally include a regulatory sequence preceding(5′ non-coding sequences) and following (3′ non-coding sequences) thecoding sequence of the nucleic acid. In some embodiments, a “gene” doesnot include regulatory sequences preceding and following the codingsequence.

In one embodiment, the gene is a heterologous gene. In anotherembodiment, the nucleic acid is a heterologous nucleic acid. As usedherein, the terms “heterologous gene” or “heterologous nucleic acid”refer to a gene or a nucleic acid sequence present in a recombinantcell, e.g., bacterium, that is not normally found in the wild-type cell,e.g., bacterium, in nature. In some embodiments, the heterologous geneor heterologous nucleic acid is exogenously introduced into a givencell. In some embodiments, a heterologous gene may include a gene, orfragment thereof, introduced into a non-native host cell. In someembodiments, the term “heterologous gene” includes a second copy of anative gene, or fragment thereof, that has been introduced into the hostcell in addition to the corresponding native gene. A heterologousnucleic acid may also include, in some embodiments, a gene sequence thatis naturally-found in a given cell but which has been modified, e.g., byregulation by a different promoter sequence, to expresses an unnaturalamount of the nucleic acid and/or the polypeptide which it encodes;and/or two or more nucleic acid sequences that are not found in the samerelationship to each other in nature.

As used herein, the term “endogenous gene” refers to a native gene thatis present in its natural location in the genome of an organism (e.g., abacterial chromosome).

A “promoter” as used herein, refers to a nucleic acid sequence that iscapable of controlling the expression of a coding sequence or gene. Apromoter may comprise one or more specific transcriptional regulatorysequences to further enhance expression and/or to alter the spatialexpression and/or temporal expression of a nucleic acid. For example, apromoter may include one or more nucleic acids that are specificallyrecognized by a transcriptional activator protein (e.g., an enhancerelement), a transcriptional repressor protein, a polymerase, and thelike. The term “operably linked,” as used herein, means that expressionof a nucleic acid sequence is under the control of a promoter with whichit is spatially connected. A promoter may be positioned 5′ (upstream) ofthe nucleic acid sequence under its control. The distance between thepromoter and a nucleic acid sequence to be expressed may beapproximately the same as the distance between that promoter and thenative nucleic acid sequence it controls. As is known in the art,variation in this distance may be accommodated without loss of promoterfunction. The nucleic acid sequences of the promoters described hereinare known in the art, and methods of operably-linking these promoters toa gene (e.g., a gene encoding a repressor) are known in the art.

In some embodiments, the promoter for use as described herein may beregulated directly or indirectly by a sugar. For example, in someembodiments, the promoter is responsive to the level of arabinose,otherwise referred to herein as an “arabinose-regulatable promoter”.Generally speaking, arabinose may be present during the in vitro growthof a bacterium, while typically absent from host tissue. In oneembodiment, the promoter is derived from an araC-P_(araBAD) system fromEscherichia coli. The araC P_(araBAD) system is a tightly regulatedexpression system, which has been shown to work as a strong promoterinduced by the addition of low levels of arabinose. The araC-araBADpromoter is a bidirectional promoter controlling expression of thearaBAD nucleic acid sequences in one direction, and the araC nucleicacid sequence in the other direction.

For convenience, the portion of the araC-araBAD promoter that mediatesexpression of the araBAD nucleic acid sequences, and which is controlledby the araC nucleic acid sequence product, is referred to herein asP_(araBAD). For use as described herein, a cassette with the araCnucleic acid sequence and the araC-araBAD promoter may be used. Thiscassette is referred to herein as araC P_(araBAD). The AraC protein isboth a positive and negative regulator of P_(araBAD). In the presence ofarabinose, the AraC protein is a positive regulatory element that allowsexpression from P_(araBAD). In the absence of arabinose, the AraCprotein represses expression from P_(araBAD). Other enteric bacteriacontain arabinose regulatory systems homologous to the araC-araBADsystem from E. coli, including, for example, S. Typhimurium. Forexample, the E. coli AraC protein only activates E. coli P_(araBAD) (inthe presence of arabinose) and not S. Typhimurium P_(araBAD). Thus, anarabinose regulated promoter may be used in a recombinant bacterium thatpossesses a similar arabinose operon, without substantial interferencebetween the two, if the promoter and the operon are derived from twodifferent species of bacteria. Generally speaking, the concentration ofarabinose necessary to induce expression is typically less than about 2%(w/w) in a culture media. In some embodiments, the concentration is lessthan about 1.5%, 1%, 0.5%, 0.2%, 0.1%, or 0.05% (w/w) in a culturemedia. In other embodiments, the concentration is 0.05% or below, e.g.about 0.04%, 0.03%, 0.02%, or 0.01% (w/w). In an exemplary embodiment,the concentration is about 0.05% (w/w) in a culture media.

In other embodiments, the promoter may be responsive to the level ofmaltose in the environment, otherwise referred to herein as a“maltose-regulatable promoter”. In some embodiments, the recombinantbacteria described herein are cultured in a medium comprising maltose.The malT gene encodes MalT, a positive regulator of fourmaltose-responsive promoters (P_(PQ), P_(EFG), P_(KBM), and P_(S)). Thecombination of malT and a mal promoter creates a tightly regulatedexpression system that has been shown to work as a strong promoterinduced in the presence of maltose. Unlike the araC-P_(araBAD) system,malT expression is regulated by a promoter (i.e., P_(T)) that isfunctionally unrelated to the other mal promoters. P_(T) is notregulated by MalT. The malEFG-malKBM promoter is a bidirectionalpromoter that controls expression of the malKBM nucleic acid sequencesin one direction, and the malEFG nucleic acid sequences in the otherdirection. For convenience, the portion of the malEFG-malKBM promoterthat mediates expression of the malKBM nucleic acid sequence, and whichis controlled by MalT, is referred to herein as P_(KBM), and the portionof the malEFG-malKBM promoter that mediates expression of the malEFGnucleic acid sequence, and which is controlled by MalT, is referred toherein as P_(EFG). Full induction of P_(KBM) requires the presence ofthe MalT binding sites of P_(EFG). For use in the vectors and systemsdescribed herein, a gene cassette comprising a nucleic acid sequenceencoding MalT and a mal promoter may be used. This gene cassette isreferred to herein as malT-P_(mal). In the presence of maltose, the MalTis a positive regulatory element that allows for expression mediated byP_(mal). Generally speaking, the concentration of maltose necessary toinduce expression is typically less than about 1% (w/w) in a culturemedia. In some embodiments, the concentration is less than about 1.0%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3% 0.2%, 0.1%, or 0.05% (w/w) in aculture media. In other embodiments, the concentration is 0.05% orbelow, e.g. about 0.04%, 0.03%, 0.02%, or 0.01% (w/w). In an exemplaryembodiment, the concentration is about 0.2% to about 0.4% (w/w) in aculture media.

In still other embodiments, the promoter used herein is responsive tothe level of rhamnose in the environment, otherwise referred to hereinas a “rhamnose-regulatable promoter”. Analogous to the araC-P_(araBAD)system described above, the rhaRS-P_(rhaB) activator-promoter system istightly regulated by rhamnose. Expression from the rhamnose promoter(P_(rha)) is induced to high levels in the presence of rhamnose. In someembodiments, the bacteria are cultured in the presence of rhamnose.Rhamnose is commonly found in bacteria but rarely found in humansubjects. The rhaBAD operon is controlled by the P_(rhaBAD) promoter.This promoter is regulated by two activators, RhaS and RhaR, and thecorresponding nucleic acid sequences belong to one transcription unitthat is located in the opposite direction of the rhaBAD nucleic acidsequences. In the presence of L-rhamnose, RhaR binds to the P_(rhaRS)promoter and activates the production of RhaR and RhaS. RhaS togetherwith L-rhamnose, in turn, bind to the P_(rhaBAD) and the P_(rhaT)promoters and activates the transcription of the structural nucleic acidsequences. Full induction of the arabinose, maltose and rhamonseregulated promoters described herein requires binding of the Crp-cAMPcomplex, which is a key regulator of catabolite repression.

Although both L-arabinose and L-rhamnose act directly as inducers of theexpression of regulons that mediate their catabolism, importantdifferences exist in regard to the regulatory mechanisms. L-Arabinoseacts as an inducer with the activator AraC in the positive control ofthe arabinose regulon. However, the L-rhamnose regulon is subject to aregulatory cascade, and is therefore subject to even tighter controlthan the araC-P_(araBAD) system. L-Rhamnose acts as an inducer with theactivator RhaR for synthesis of RhaS, which in turn acts as an activatorin the positive control of the rhamnose regulon. In the presentdisclosure, rhamnose may be used to interact with the RhaR protein andthen the RhaS protein may activate transcription of a nucleic acidsequence operably-linked to the P_(rhaBAD) promoter.

In still other embodiments, the promoter may be responsive to the levelof xylose in the environment, referred to herein as a“xylose-regulatable promoter”. Generally, xylose concentrations ofbetween 0.0002% to 0.63% (w/w) in the environment activate theexpression of a xylose inducible promoter described herein (see, e.g.,Bhavsar et al. (2001) App. Environ. Microbiol. 67(1): 403-10(34)). ThexylR-P_(xylA) system is another well-established inducibleactivator-promoter system. Xylose induces xylose-specific operons (e.g.,xylE, xylFGHR, and xylAB) that are regulated by XylR and the cyclicAMP-Crp system. The XylR protein serves as a positive regulator bybinding to two distinct regions of the xyl nucleic acid sequencepromoters. As with the araC-P_(araBAD) system described above, thexylR-P_(xylAB) and/or xylR-P_(xylFGH) regulatory systems may be used. Inthese embodiments, xylose interacting with the XylR protein activatestranscription of nucleic acid sequences operably-linked to either of thetwo P_(xyl) promoters.

As used herein, the term “exogenous” refers to a substance (e.g., anucleic acid or polypeptide) present in a cell other than its nativesource. The term exogenous can refer to a nucleic acid or a protein thathas been introduced by a process involving the hand of man into abiological system such as a cell or organism in which it is not normallyfound or in which it is found in undetectable amounts. A substance canbe considered exogenous if it is introduced into a cell or an ancestorof the cell that inherits the substance. In contrast, the term“endogenous” refers to a substance that is native to the biologicalsystem or cell.

A “pharmaceutical composition,” as used herein, refers to a compositioncomprising an active ingredient (e.g., a recombinant bacterium describedherein) with other components such as a physiologically suitable carrierand/or excipient.

As used herein, the term “pharmaceutically acceptable carrier” or a“pharmaceutically acceptable excipient” refers to apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline (e.g.,phosphate-buffered saline (PBS)); (18) Ringer's solution; (19) ethylalcohol; (20) pH buffered solutions; (21) polyesters, polycarbonatesand/or polyanhydrides; (22) bulking agents, such as polypeptides andamino acids (23) serum component, such as serum albumin, HDL and LDL;(24) C₂-C₁₂ alcohols, such as ethanol; and (25) other non-toxiccompatible substances employed in pharmaceutical formulations. Wettingagents, coloring agents, release agents, coating agents, disintegratingagents, binders, sweetening agents, flavoring agents, perfuming agents,protease inhibitors, plasticizers, emulsifiers, stabilizing agents,viscosity increasing agents, film forming agents, solubilizing agents,surfactants, preservative and antioxidants can also be present in theformulation. The terms such as “excipient”, “carrier”, “pharmaceuticallyacceptable excipient” or the like are used interchangeably herein.

A “plasmid” or “vector” includes a nucleic acid construct designed fordelivery to a host cell or transfer between different host cells. Thenucleic acid incorporated into the plasmid can be operatively linked toan expression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. The terms“protein” and “polypeptide” as used herein refer to both largepolypeptides and small peptides. The terms “protein” and “polypeptide”are used interchangeably herein when referring to a gene product andfragments thereof. Thus, exemplary polypeptides or proteins include geneproducts, naturally occurring proteins, homologs, orthologs, paralogs,fragments and other equivalents, variants, fragments, and analogs of theforegoing.

A “nucleic acid” or “nucleic acid sequence” may be any molecule,preferably a polymeric molecule, incorporating units of ribonucleicacid, deoxyribonucleic acid or an analog thereof. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one nucleic acid strand of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the nucleic acid can be DNA. Inanother aspect, the nucleic acid can be RNA. Suitable nucleic acidmolecules are DNA, including genomic DNA or cDNA. Other suitable nucleicacid molecules are RNA, including mRNA, rRNA, and tRNA.

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare very well established and include, for example, those disclosed byWalder et al. (35); Bauer et al. (36); Craik (37); Smith et al. (38);and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties. Any cysteine residue notinvolved in maintaining the proper conformation of the polypeptide alsocan be substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) can be added to the polypeptide to improve itsstability or facilitate oligomerization.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

As used herein, the term “host cell” refers to a cell in an organism towhich the recombinant bacterium is being administered in order to, forexample, induce an immune response. In one embodiment, a host is a bird,equine, or human and a host cell refers, respectively, to a bird cell,an equine cell, or a human cell.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

The articles “a” and “an,” as used herein, should be understood to mean“at least one,” unless clearly indicated to the contrary.

The phrase “and/or,” when used between elements in a list, is intendedto mean either (1) that only a single listed element is present, or (2)that more than one element of the list is present. For example, “A, B,and/or C” indicates that the selection may be A alone; B alone; C alone;A and B; A and C; B and C; or A, B, and C. The phrase “and/or” may beused interchangeably with “at least one of” or “one or more of” theelements in a list.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

I. Recombinant Bacteria

The present disclosure provides, in some embodiments, a recombinantbacterium capable of regulated expression of at least one nucleic acidsequence encoding an antigen of interest. The recombinant bacteriumdescribed herein is particularly effective in eliciting an immuneresponse (e.g., protective immunity) against the antigen of interestbecause the bacterium comprise multiple recombinant regulatory systemsthat permit the bacterium to replicate upon administration and tocolonize lymphoid tissues in a subject in order to elicit potent immuneresponses. However, after multiple replication cycles in vivo, thebacterium ultimately exhibits an attenuated phenotype which allows forsafe administration to a subject, for example as a vaccine composition.The recombinant regulatory systems of the bacteria described hereindepend, in part, on multiple genetic regulatory elements that areresponsive to one or more sugars (e.g., arabinose, rhamnose, mannose,maltose, xylose, and galactose) that not available to the bacterium invivo. Thus, using the phenotype of the recombinant bacteria describedherein can be altered upon administration to a subject. In someembodiments, the subject is administered one or more sugars before,after or concurrently with the administration of a recombinant bacteriumdescribed herein in order to activate and/or repress a sugar-responsiveregulatory system of the bacteria. In some embodiments, the recombinantbacterium described herein comprises at least three regulatory systems,each dependent on a different sugar, which facilitates initial invasionof a host cell in the subject, delayed attenuation, and improvedimmunogenicity.

In some embodiments, the recombinant bacterium described herein can beregulated for delayed attenuation in vivo. In some embodiments, therecombinant bacterium described herein is capable of regulated delayedexpression of a nucleic acid encoding an antigen of interest. In someembodiments, the recombinant bacterium described herein exhibitsregulated production of Generalized Modules for Membrane Antigens(GMMA), or outer membrane vesicles, in vivo, which may lead to enhancedproduction of conserved outer membrane proteins present in thebacterium, and ultimately improved immunogenicity. In some embodiments,the recombinant bacterium described herein is capable of both regulatedexpression of at least one nucleic acid encoding at least one antigen ofinterest and regulated attenuation. In some embodiments, the recombinantbacterium described herein is capable of both regulated expression of atleast one nucleic acid encoding at least one antigen of interest andregulated production of GMMA, or outer membrane vesicles, in vivo. Insome embodiments, the recombinant bacterium described herein is capableof both regulated production of GMMA, or outer membrane vesicles, invivo, and regulated attenuation. In some embodiments, the recombinantbacterium described herein is capable of regulated expression of atleast one nucleic acid encoding at least one antigen of interest,regulated attenuation, and regulated production of GMMA, or outermembrane vesicles, in vivo. In some embodiments, each of theseproperties is directly or indirectly regulated by the abundance of atleast one sugar (e.g., arabinose, rhamnose, mannose, xylose, maltose,and galactose).

In some embodiments, the bacterium described herein is a Gram negativebacterium. In some embodiments, the bacterium is a pathogenic bacterium.In some embodiments, the bacterium is an avirulent bacterium. In someembodiments, the bacterium belongs to the Enterobaceteriaceae. In someembodiments, the bacterium belongs to a genus selected from:Alterococcus, Aquamonas, Aranicola, Arsenophonus, Brenneria, Budvicia,Buttiauxella, Candidatus Phlomobacter, Cedeceae, Citrobacter,Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Hafnia,Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella, Morganella,Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Plesiomonas,Pragia, Proteus, Providencia, Rahnella, Raoultella, Salmonella,Samsonia, Serratia, Shigella, Sodalis, Tatumella, Trabulsiella,Wigglesworthia, Xenorhbdus, or Yersinia, Yokenella. In some embodiments,the bacterium is a pathogenic species of Enterobaceteriaceae. In someembodiments, the bacterium is selected from the group consisting ofEscherichia coli, Shigella, Edwardsiella, Salmonella, Citrobacter,Klebsiella, Enterobacter, Serratia, Proteus, Morganella, Providencia andYersinia. In some embodiments, the bacterium is of the genus Salmonella.In some embodiments, the bacterium is of the genus Yersinia. In someembodiments, the bacterium is of the genus Edwardsiella. In someembodiments, the bacterium is of a genus, species, or strain commonlyused as a live or attenuated vaccine.

Some embodiments of the instant disclosure comprise a species orsubspecies of the Salmonella genera (e.g., S. enterica or S. bongori).For instance, the recombinant bacterium may be a Salmonella entericaserovar, including, for example, Paratyphi A, Enteritidis, Typhi, andTyphimurium. In some embodiments, the recombinant bacterium is of theserovar S. Typhimurium, S. Typhi, S. Paratyphi, S. Gallinarum, S.Enteritidis, S. Choleraesius, S. Arizonae, S. Newport, S. Heidelberg, S.Infantis, S. Cholerasiuis, or S. Dublin.

A recombinant bacterium derived from Salmonella may be particularlysuited to use as a vaccine. For example, oral infection of a host with aSalmonella strain typically leads to colonization of the gut-associatedlymphoid tissue (GALT) or Peyer's patches, which leads to the inductionof a generalized mucosal immune response to the recombinant bacterium.Further penetration of the bacterium into the mesenteric lymph nodes,liver and spleen may augment the induction of systemic and cellularimmune responses directed against the bacterium. Thus, the use ofrecombinant Salmonella for oral immunization stimulates all threebranches of the immune system, which is particularly important forimmunizing against infectious disease agents that colonize on and/orinvade through mucosal surfaces. In some embodiments, the recombinantbacterium described herein is used to induce an immune response inpoultry (e.g., as a vaccine). When used in poultry, the recombinantbacterium may be administered by course spray and thereby inoculate theconjunctiva-associated lymphoid tissue (CALT) via eye exposure, thenasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoidtissue (BALT) via respiratory exposure and the GALT via oral exposure.In some embodiments, the recombinant bacterium described herein isadministered to newly-hatched chicks.

A. Attenuation

In some embodiments, the recombinant bacterium described herein ismodified such that the expression of one or more genes, e.g., virulencegenes, can be regulated in a sugar-responsive manner. In someembodiments, one or more endogenous genes, e.g., virulence genes, aredeleted from the bacterial chromosome. In some embodiments, the deletionis a partial deletion of the endogenous gene. In some embodiments, thedeletion is a full-length deletion of the endogenous gene. In someembodiments, the gene, e.g., virulence gene, is genetically-altered toprevent transcription and/or translation of the gene encoding theprotein. In some embodiments, the endogenous gene is genetically alteredto insert a transcriptional terminator in the open reading frame of thegene. In some embodiments, a regulatory region of the gene, e.g.,virulence gene, is genetically-modified to alter (e.g., decrease) theexpression of the gene. In some embodiments, the promoter of a gene,e.g., virulence gene, is altered to include one or more regulatoryelements (e.g., a sugar-responsive promoter).

In some embodiments, the recombinant bacterium described herein ismodified to comprise a nucleic acid comprising a gene. In someembodiments, the recombinant bacterium is modified to comprise a nucleicacid comprising a gene, whereby an endogenous copy of the gene in thebacterial chromosome has been altered and/or deleted. In someembodiments, the nucleic acid comprises a gene that is at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to an endogenous gene in the bacterial chromosome thathas been deleted and/or altered. In some embodiments, the nucleic acidcomprises a gene that is at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% homologous to anendogenous gene in the bacterial chromosome that has been deleted and/oraltered. In some embodiments, the nucleic acid comprises a gene from abacterial species, subspecies, serovar, or strain that is different thanthe bacterial species of the recombinant bacterium.

In some embodiments, the nucleic acid comprises a gene from a bacterialspecies, subspecies, serovar, or strain that is the same as thebacterial species of the recombinant bacterium. In some embodiments, thenucleic acid comprises a gene that is operably-linked to a regulatablepromoter (e.g., a sugar-regulatable promoter). In some embodiments, thenucleic acid comprises a gene that is operably-linked to arhamnose-regulatable promoter, a xylose-regulatable promoter, agalactose-regulatable promoter, an arabinose-regulatable promoter, amannose-regulatable promoter, or a maltose-regulatable promoter. In someembodiments, the nucleic acid comprising the gene is located in aplasmid in the bacterium. In some embodiments, the nucleic acidcomprising the gene is located in the bacterial chromosome. In someembodiments, the nucleic acid comprising the gene is located at thechromosomal locus corresponding to the locus of an endogenous gene thathas been deleted or altered in the bacterial chromosome. In someembodiments, the nucleic acid is codon-optimized (e.g., to improveexpression of the nucleic acid in the recombinant bacterium).

1. O-Antigen Synthesis Genes

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous O-antigen synthesis gene. In some embodiments, therecombinant bacterium comprises a deletion in an endogenous O-antigenligase gene. In some embodiments, the deletion is a partial deletion ofthe endogenous O-antigen ligase gene. In some embodiments, the deletionis a full-length deletion of the endogenous O-antigen ligase gene. Insome embodiments, the endogenous O-antigen ligase gene is geneticallyaltered to insert a transcriptional terminator in the open reading frameof the gene. In some embodiments, a regulatory region of the endogenousO-antigen ligase gene is genetically-modified to alter (e.g., decrease)the expression of the gene. In some embodiments, the promoter of aendogenous O-antigen ligase gene is altered to include one or moreregulatory elements (e.g., a sugar-responsive promoter). In someembodiments, the promoter of a endogenous O-antigen ligase gene isaltered to increase the spacing between the Shine-Delgarno sequence andthe start codon of the gene. In some embodiments, the promoter of aendogenous O-antigen ligase gene is altered to decrease the spacingbetween the Shine-Delgarno sequence and the start codon of the gene. Insome embodiments, the Shine-Delgarno (SD) sequence, the start codon, thesecond codon and/or third codons of the O-antigen ligase gene is alteredto increase the frequency of adenine nucleobases in order to enhance thetranslation efficiency of the gene. In some embodiments, theShine-Delgarno (SD) sequence, the start codon, the second codon and/orthird codons of the O-antigen ligase gene is altered to reduce thefrequency of adenine nucleobases in order to decrease the translationefficiency of the gene. In some embodiments, the O-antigen ligase geneis waaL (also known as rfaL). The O-antigen ligase WaaL is necessary toligate polysaccharide to the lipid A-LPS core moiety. Deletion of waaLresults in an intact lipid A-LPS core with no O-antigen or individualsugars attached to it. In some embodiments, the O-antigen ligase gene isselected from the group consisting of waaG (also known as rfaG), waal(also known as rfal), rfaH, waaJ (also known as rfaJ), wbaP (also knownas rfbP), wzy (also known as rfc), waaP, waaQ, waaF, waaP, waaC, andwaaA.

In some embodiments, the recombinant bacterium described herein ismodified to comprise a nucleic acid comprising an O-antigen ligase gene.In some embodiments, the nucleic acid comprising an O-antigen ligasegene is located on a plasmid in the bacterium. In some embodiments, thenucleic acid comprising an O-antigen ligase gene is located on achromosome of the bacterium. In some embodiments, the nucleic acidcomprising an O-antigen ligase gene is located at the chromosomal locuscorresponding to the locus of an endogenous O-antigen ligase gene thathas been deleted or altered in the bacterial chromosome. In someembodiments, the recombinant bacterium is modified to comprise a nucleicacid comprising an O-antigen ligase gene, whereby an endogenous copy ofthe gene in the bacterial chromosome has been altered and/or deleted. Insome embodiments, the nucleic acid comprises a Salmonella O-antigenligase gene.

The nucleic acid sequence of an exemplary Salmonella waaL gene isprovided below:

(SEQ ID NO: 1) atgctaaccacatcattaacgttaaataaagagaaatggaagccgatctggaataaagcgctggtttttctttttgttgccacgtattttctggatggtattacgcgttataaacatttgataatcatacttatggttatcaccgcgatttatcaggtctcacgctcaccgaaaagtttcccccctcttttcaaaaatagcgtattttatagcgtagcagtattatcattaatccttgtttattccatactcatatcgccagatatgaaagaaagtttcaaggaatttgaaaatacggtactggagggcttcttattatatactttattaattcccgtactattaaaagatgaaacaaaagaaacggttgcgaaaatagtacttttctcctttttaacaagtttaggacttcgctgccttgcagagagtattctgtatatcgaggactataataaagggattatgccattcataagctatgcgcatcgacatatgtccgattccatggttttcttatttccagcattattgaatatttggctgtttagaaaaaatgcaattaagttggtttttttggtgcttagcgccatctaccttttctttatcctgggaaccctatcgcgaggggcatggttggcggtgcttatagtaggtgttctgtgggcaatactgaaccgccaatggaagttaataggagttggtgccattttattagccattatcggcgctttggttatcactcaacataataacaaaccagacccagaacatttactgtataaattacagcagacagatagctcatatcgttatactaacggaacccagggcaccgcgtggatactgattcaggaaaacccgatcaagggctacggctatggtaatgatgtgtatgatggtgtttataataaacgcgttgtcgattatccaacgtggacctttaaagaatctatcggtccgcataataccattctgtacatctggtttagtgcaggcatattgggtctggcgagcctggtctatttatatggcgctatcatcagggaaacagccagctctaccctcaggaaagtagagataagcccctacaatgctcatctcttgctatttttatctttcgtcggtttttatatcgttcgtggcaattttgaacaggtcgatattgctcaaattggtatcattaccggttttctgctggcg ctaagaaatagataa. 

The amino acid sequence of the WaaL protein encoded by the nucleic acidof SEQ ID NO: 1 is provided below:

(SEQ ID NO: 2) MLTTSLTLNKEKWKPIWNKALVFLFVATYFLDGITRYKHLIIILMVITAIYQVSRSPKSFPPLFKNSVFYSVAVLSLILVYSILISPDMKESFKEFENTVLEGFLLYTLLIPVLLKDETKETVAKIVLFSFLTSLGLRCLAESILYIEDYNKGIMPFISYAHRHMSDSMVFLFPALLNIWLFRKNAIKLVFLVLSAIYLFFILGTLSRGAWLAVLIVGVLWAILNRQWKLIGVGAILLAIIGALVITQHNNKPDPEHLLYKLQQTDSSYRYTNGTQGTAWILIQENPIKGYGYGNDVYDGVYNKRVVDYPTWTFKESIGPHNTILYIWFSAGILGLASLVYLYGAIIRETASSTLRKVEISPYNAHLLLFLSFVGFYIVRGNFEQVDIAQIG IITGFLLALRNR. 

In some embodiments, the nucleic acid comprises a Salmonella waaL gene(provided as SEQ ID NO: 1). In some embodiments, the nucleic acidcomprises a waaL gene, wherein the waaL gene comprises a nucleic acidsequence that is at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the nucleic acid sequenceof SEQ ID NO: 1. In some embodiments, the nucleic acid comprises a waaLgene, wherein the waaL gene comprises a nucleic acid sequence that is atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding an O-antigen ligase, wherein said O-antigen ligase comprises anamino acid sequence that is at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to the aminoacid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acidcomprises a nucleic acid sequence encoding an O-antigen ligase, whereinsaid O-antigen ligase comprises an amino acid sequence that is at least75%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid comprises an O-antigen ligase genefrom a bacterial species, subspecies, serovar, or strain that isdifferent than the bacterial species of the recombinant bacterium. Insome embodiments, the nucleic acid comprises an O-antigen ligase genefrom a bacterial species, subspecies, serovar, or strain that is thesame as the bacterial species of the recombinant bacterium.

In some embodiments, the nucleic acid comprises an O-antigen ligase genethat is operably-linked to a regulatable promoter (e.g., asugar-regulatable promoter). In some embodiments, the nucleic acidcomprises an O-antigen ligase gene (e.g., waaL) that is operably-linkedto a sugar-regulatable promoter. Advantageously, recombinant bacterialstrains comprising a nucleic acid comprising an O-antigen ligase gene(e.g., waaL) that is operably linked to a sugar regulatable promoterwill synthesize normal LPS in the presence of the sugar (e.g., rhamnose)in vitro, but will form rough LPS in vivo due to the absence of thesugar that activates the promoter and therefore, the expression of theO-antigen ligase. Without wishing to be bound by any particular theory,using this strategy, the bacterium will expose conserved LPS coreoligosaccharide and have enhanced production of conserved outer membraneproteins (OMPs; e.g., porins) which may lead to improved immunogenicityand aid in the production of a cross-protective immune response againstan antigen of interest synthesized in the bacterium in vivo. In someembodiments, the sugar regulatable promoter exhibits increased activity(e.g., increased transcription) in the presence of a specific sugar anddecreased activity in the absence of a sugar. In some embodiments, thenucleic acid comprises an O-antigen ligase gene that is operably-linkedto a rhamnose-regulatable promoter (e.g., a sugar-regulatable promoter).In some embodiments, the nucleic acid comprises an O-antigen ligase genethat is operably-linked to an arabinose-regulatable promoter (e.g., asugar-regulatable promoter). In some embodiments, the use of arhamnose-regulatable promoter (e.g., rhaSR P_(rhaBAD)) may be preferableto an arabinose-regulatable promoter because a relatively higherconcentration is required to activate an arabinose-regulatable promoteras compared to a rhamnose-regulatable promoter (see, e.g., Giacalone etal. (2006) BioTechniques 40(3): 355-366 (39), the entire contents ofwhich are incorporated herein by reference). In some embodiments, therecombinant bacterium comprises the mutation ΔwaaL/ΔpagL::TT rhaSRP_(rhaBAD) waaL.

2. Lipid A Deacylase Genes

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous lipid A deacylase gene. In some embodiments, the deletionis a partial deletion of the endogenous lipid A deacylase gene. In someembodiments, the deletion is a full-length deletion of the endogenouslipid A deacylase gene. In some embodiments, the endogenous lipid Adeacylase gene is genetically altered to insert a transcriptionalterminator in the open reading frame of the gene. In some embodiments, aregulatory region of the endogenous lipid A deacylase gene isgenetically-modified to alter (e.g., decrease) the expression of thegene. In some embodiments, the promoter of an endogenous lipid Adeacylase gene is altered to include one or more regulatory elements(e.g., a sugar-responsive promoter). In some embodiments, the lipid Adeacylase gene ispagL. Bacterial comprising a deletion of the lipid Adeacylase gene pagL have been found to produced increased amounts ofouter membrane vesicles (see, e.g., Elhenawy et al. (2016) mBio 7(4):e00940-16 (40)). Deletion of the pagL gene of Salmonella does not impairbacterial virulence (see, e.g., Man et al. Proc. Nat'l. Acad. Sci. USA111: 7403-8 (41)). Without wishing to be bound by any particular theory,in some embodiments, the recombinant bacterium described herein compriseone or more genetic modifications which results in increasedvesiculation (i.e., increased vesicle production) which may beparticularly advantageous in inducing an immune response in the hostagainst an antigen of interest that is expressed by the bacterium.

3. Phosphomannose Isomerase Genes

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous phosphomannose isomerase gene. Phosphomannose isomerase,also known as mannose-6 phosphate isomerase, catalyzes the reversibleinterconversion of fructose 6-phosphate to mannose 6-phosphate. Mannose6-phosphate is then converted to GDP-mannose and used for the synthesisof O-antigen side chains. Bacteria with deletions of the phosphomannoseisomerase gene pmi are not mannose sensitive and are partiallyattenuated (see, e.g., Collins et al. (1991) Infect. Immun. 59(3):1079-85 (42)). Thesepmi mutants synthesize wild-type levels of LPSO-antigen side chains when grown in media containing mannose, and areboth attenuated but highly immunogenic (see, e.g., Curtiss et al. (2007)“Induction of host immune responses using Salmonella-vectored vaccines.”In: Brogden K A, Minion F C, Cornick N, Stanton T B, Zhang Q, Nolan L K,Wannemuehler M J, ed. Virulence Mechanisms of Bacterial Pathogens. 4thed. Washington D.C.: ASM Press (43)). In some embodiments, the deletionof the endogenous phosphoisomerase gene is a partial deletion. In someembodiments, the deletion of the endogenous phosphomannose isomerasegene is a full-length deletion. In some embodiments, the endogenousphosphomannose isomerase gene is genetically altered to insert atranscriptional terminator in the open reading frame of the gene. Insome embodiments, a regulatory region of the endogenous phosphomannoseisomerase gene is genetically-modified to alter (e.g., decrease) theexpression of the phosphomannose isomerase gene. In some embodiments,the promoter of an endogenous phosphomannose isomerase gene is alteredto include one or more regulatory elements (e.g., a sugar-responsivepromoter). In some embodiments, the phosphomannose isomerase gene ispmi.

In some embodiments, the bacterium comprises a deletion of apmi gene. Insome embodiments, the bacterium comprises a Δpmi-2426 mutation. Abacterium comprising a Δpmi-2426 mutation, grown in the presence ofmannose, is capable of synthesizing a complete LPS O-antigen.Non-phosphorylated mannose, which is the form required for bacterialuptake, is unavailable in vivo. Hence, a bacterium comprising aΔpmi-2426 mutation loses the ability to synthesize LPS O-antigenserotype specific side chains in vivo and the number of O-antigen sidechains attached to the LPS core decreases by about half after each celldivision in vivo. The LPS that is synthesized comprises a core structurethat is substantially similar across all Salmonella enterica serotypesexcept S. Arizona. This results in a bacterium that is capable ofeliciting an immune response against at least two Salmonella serotypeswithout substantially inducing an immune response specific to theserotype of the bacterial vector. In some embodiments, the bacterium iscapable of eliciting an immune response against all Salmonella serotypeswithout substantially inducing an immune response specific to theserotype of the bacterial vector.

A recombinant bacterium described herein that comprises a deletion inapmi mutation may also comprise other mutations that ensure that mannoseavailable to the bacterium during in vitro growth is used for LPSO-antigen synthesis. For instance, a bacterium may comprise aΔ(gmd-fcl)-26 mutation. This mutation deletes two nucleic acid sequencesthat encode enzymes for conversion of GDP-mannose to GDP-fucose,ensuring that mannose available to the bacterium during in vitro growthis used for LPS O-antigen synthesis and not colanic acid production.Similarly, a bacterium may comprise the Δ(wcaM-wza)-8 mutation, whichdeletes all 20 nucleic acid sequences necessary for colanic acidproduction, and also precludes conversion of GDP-mannose to GDP-fucose.

4. UDP-Galactose Epimerase Genes

UDP-Gal is the precursor for the assembly of the LPS O-antigen sidechains, the LPS outer core, for colanic acid and other polysaccharidepolymers having galactose as a constituent (44). UDP-Gal is synthesizedby conversion of glucose-1-P to UDP-Glu by the enzyme glucose-1-Puridylyltransferase encoded by the galU gene with UDP-Glu converted toUDP-Gal by the enzyme UDP-galactose epimerase encoded by the galE gene(45, 46). Strains grown in the presence of galactose can synthesizeUDP-Gal by a different pathway in which galactose after uptake isconverted to galactose-1-P by galactose kinase encoded by the galK genewhich in turn is converted to UDP-Gal by the enzyme UDP-Gal transferaseencoded by the galT gene (45). Strains with a galE mutation are unableto synthesize LPS outer core and LPS O-antigen unless galactose issupplied in the growth medium (47). Because of these facts andproperties Salmonella strains with galE mutations can synthesize LPSwhen grown with galactose and are invasive to colonize lyphoid tissues,but loose this ability in vivo due to the unavailability of freegalactose such that they gradualy loose LPS components as they multiplyin the infected or immunized animal host. Just like pmi mutants, theygradually become attenuated due to increasing susceptibility tocomplement-mediated cytotoxicity and enhanced phagocytosis and killingmy macrophages. However, the supply of galactose to such galE mutantscan lead to cell death by lysis since the accumulation of Gal-1-P andUDP-Gal is toxic (30, 48, 49). Because of this, growth of galE mutantsin the presence of galactose selects for mutations in genes forgalactose uptake or in the galK and galT genes so that toxic productsare not synthesized. Unfortunately, such galactose-resistant mutants areno longer able to make LPS and are totally attenuated, non-invasive andnon-immunogenic (30, 50). To circumvent these problems to enable use ofgalE mutations in Salmonella vaccine strains, we have devised a means togenerate galE mutants with the potential for reversible synthesis of LPSdependent on the presence or absence of galactose that are resistant togalactose with no selection of mutants unable to synthesize UDP-Gal forLPS synthesis.

5. Iron Acquisition Regulatory Genes

In some embodiments, the recombinant bacterium comprises a deletion inthe endogenous promoter P_(fur), which regulates the expression of thefur gene. Fur represses the transcription of genes involved in ironacquisition in the presence of free iron. When iron concentrationsbecome low in the bacterium, Fur ceases to be synthesized which leads tothe constitutive expression of genes encoding iron acquisition proteins(e.g., iron-regulated outer membrane proteins (IROMPs). In someembodiments, the deletion is a partial deletion of the endogenousP_(fur) promoter. In some embodiments, the deletion is a full-lengthdeletion of the endogenous P_(fur) promoter. In some embodiments, theendogenous P_(fur) promoter is genetically-modified to alter (e.g.,decrease) the expression of the fur gene. In some embodiments, theendogenous P_(fur) promoter is genetically altered to comprise atranscriptional terminator.

In some embodiments, the recombinant bacterium comprises a nucleic acidcomprising a fur gene (e.g., a fur gene from the same bacterial speciesas the recombinant bacterium).

In some embodiments, the nucleic acid comprising a fur gene is locatedon a plasmid in the bacterium. In some embodiments, the nucleic acidcomprising a fur gene is located on a chromosome of the bacterium. Insome embodiments, the nucleic acid comprising a fur gene is located atthe chromosomal locus corresponding to the locus of an endogenous furgene that has been deleted or altered in the bacterial chromosome. Insome embodiments, the recombinant bacterium is modified to comprise anucleic acid comprising a fur gene, whereby an endogenous copy of thefiur gene in the bacterial chromosome has been altered and/or deleted.

The nucleic acid sequence of an exemplary Salmonella fur gene isprovided below:

(SEQ ID NO: 3) atgactgacaacaataccgcattaaagaaggctggcctgaaagtaacgcttcctcgtttaaaaattctggaagttcttcaggaaccagataaccatcacgtcagtgcggaagatttatacaaacgcctgatcgacatgggtgaagaaatcggtctggcaaccgtataccgtgtgctgaaccagtttgacgatgccggtatcgtgacccgccataattttgaaggcggtaaatccgtttttgaactgacgcaacagcatcatcacgaccatcttatctgccttgattgcggaaaagtgattgaatttagtgatgactctattgaagcgcgccagcgtgaaattgcggcgaaacacggtattcgtttaactaatcacagcctctatctttacggccactgcgctgaaggcgactgccgcgaagacgagcacgcgcacgatgac gcgactaaataa. 

The amino acid sequence of the Fur protein encoded by the nucleic acidof SEQ ID NO: 3 is provided below:

(SEQ ID NO: 4) MTDNNTALKKAGLKVTLPRLKILEVLQEPDNHEIVSAEDLYKRLIDMGEEIGLATVYRVLNQFDDAGIVTRHNFEGGKSVFELTQQHHHDHLICLDCGKVIEFSDDSIEARQREIAAKHGIRLTNHSLYLYGHCAEGDCREDEHAHD DATK. 

In some embodiments, the nucleic acid comprises a Salmonella fur gene(provided as SEQ ID NO: 3). In some embodiments, the nucleic acidcomprises a fur gene, wherein the fur gene comprises a nucleic acidsequence that is at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the nucleic acid sequenceof SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a furgene, wherein the fur gene comprises a nucleic acid sequence that is atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO:3.

In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding a Fur protein, wherein said Fur protein comprises an amino acidsequence that is at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO: 4. In some embodiments, the nucleic acid comprises a nucleicacid sequence encoding a Fur protein, wherein said Fur protein comprisesan amino acid sequence that is at least 75%, at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% homologous to the aminoacid sequence of SEQ ID NO: 4.

In some embodiments, the nucleic acid comprises a fur gene from abacterial species, subspecies, serovar, or strain that is the same asthe bacterial species of the recombinant bacterium.

In some embodiments, the nucleic acid comprises a fur gene that isoperably-linked to a regulatable promoter (e.g., a sugar-regulatablepromoter). In some embodiments, the nucleic acid comprises a fur genethat is operably-linked to a sugar-regulatable promoter. In someembodiments, the sugar regulatable promoter exhibits increased activity(e.g., increased transcription) in the presence of a specific sugar anddecreased activity in the absence of a sugar. In some embodiments, thenucleic acid comprises a fur gene that is operably-linked to arhamnose-regulatable promoter (e.g., a sugar-regulatable promoter). Insome embodiments, the nucleic acid comprises a fir gene that isoperably-linked to an arabinose-regulatable promoter (e.g., asugar-regulatable promoter). In some embodiments, thearabinose-regulatable promoter is araC P_(araBAD). In some embodiments,the recombinant bacterium comprises the mutation ΔP_(fur)::TT araCP_(araBAD) fur.

6. Colicin Uptake Genes

Salmonella spontaneously release 50 to 90 nm bleb-like particles ofouter cell wall membrane called Generalized Modules for MembraneAntigens (GMMA) or outer membrane vesicles, which constitute an enrichedsource of outer membrane-associated antigens that retain their nativeconfirmation and proper orientation. Salmonella can begenetically-modified to produce more GMMAs, or outer membrane vesicles(e.g., by deletion of a tolR gene) which can be readily purified (e.g.,by centrifugation and filtration in the absence of detergent). GMMAs, orouter membrane vesicles, contain multiple pathogen-associated molecularpatterns (PAMPS), including toll-like receptor (TLR) ligands, which mayact as self-adjuvants when eliciting immune responses. Recombinantbacteria that do not express tolR produce more GMMA, or outer membranevesicles, which may be particularly advantageous in increasing thepresentation of conserved proteins to aid in inducing, for example,antibodies cross-reactive to OMPs of other Salmonella serovars. Inaddition, without wishing to be bound by any particular theory,increased production and release of GMMA, or outer membrane vesicles,will also lead to the improved presentation of an antigen of interestthat is expressed by the recombinant bacterium as described herein. Insome embodiments, the antigen of interest is a secreted antigen.

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous gene encoding a colicin uptake protein. Two types ofcolicins have been described. Group A colicins are Tol-dependentcolicins and Group B colicins are TonB-dependent colicins (see, e.g.,Cascales et al. (2007) Microbiol. Mol. Biol. Rev. 71(1): 158-229 (51),the entire contents of which are hereby incorporated by reference). Insome embodiments, the recombinant bacterium comprises a deletion in theendogenous promoter P_(tolR), which regulates the expression of the tolRgene. This deletion will cause the endogenous tolR gene to not beexpressed by the recombinant bacterium comprising the deletion. In someembodiments, the endogenous P_(toR) promoter is genetically-modified toalter (e.g., decrease) the expression of the tolR gene. In someembodiments, the endogenous P_(tolR) promoter is genetically altered tocomprise a transcriptional terminator.

In some embodiments, the recombinant bacterium described herein ismodified to comprise a nucleic acid comprising a tolR gene. In someembodiments, the nucleic acid comprising a tolR gene is located on aplasmid in the bacterium. In some embodiments, the nucleic acidcomprising a tolR gene is located on a chromosome of the bacterium. Insome embodiments, the nucleic acid comprising a tolR gene is located atthe chromosomal locus corresponding to the locus of an endogenous a tolRthat has been deleted or altered in the bacterial chromosome. In someembodiments, the recombinant bacterium is modified to comprise a nucleicacid comprising a tolR gene, whereby an endogenous copy of the tolR genein the bacterial chromosome has been altered and/or deleted.

The nucleic acid sequence of an exemplary Salmonella tolR gene isprovided below:

(SEQ ID NO: 5) atgactgacaacaataccgcattaaagaaggctggcctgaaagtaacgcttcctcgtttaaaaattctggaagttcttcaggaaccagataaccatcacgtcagtgcggaagatttatacaaacgcctgatcgacatgggtgaagaaatcggtctggcaaccgtataccgtgtgctgaaccagtttgacgatgccggtatcgtgacccgccataattttgaaggcggtaaatccgtttttgaactgacgcaacagcatcatcacgaccatcttatctgccttgattgcggaaaagtgattgaatttagtgatgactctattgaagcgcgccagcgtgaaattgcggcgaaacacggtattcgtttaactaatcacagcctctatctttacggccactgcgctgaaggcgactgccgcgaagacgagcacgcgcacgatgacgcgactaaa taa. 

The amino acid sequence of the TolR protein encoded by the nucleic acidof SEQ ID NO: 5 is provided below:

(SEQ ID NO: 6) MTDNNTALKKAGLKVTLPRLKILEVLQEPDNHEIVSAEDLYKRLIDMGEEIGLATVYRVLNQFDDAGIVTRHNFEGGKSVFELTQQHHHDHLICLDCGKVIEFSDDSIEARQREIAAKHGIRLTNHSLYLYGHCAEGDCREDEHAHDDAT K. 

In some embodiments, the nucleic acid comprises a Salmonella tolR gene(provided as SEQ ID NO: 5). In some embodiments, the nucleic acidcomprises a tolR gene, wherein the tolR gene comprises a nucleic acidsequence that is at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the nucleic acid sequenceof SEQ ID NO: 5. In some embodiments, the nucleic acid comprises a tolRgene, wherein the tolR gene comprises a nucleic acid sequence that is atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO:5.

In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding a TolR protein, wherein said TolR protein comprises an aminoacid sequence that is at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the amino acidsequence of SEQ ID NO: 6. In some embodiments, the nucleic acidcomprises a nucleic acid sequence encoding a TolR protein, wherein saidTolR protein comprises an amino acid sequence that is at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% homologous to the nucleic acid sequence of SEQ ID NO: 6.

In some embodiments, the nucleic acid comprises a tolR gene from abacterial species, subspecies, serovar, or strain that is different thanthe bacterial species of the recombinant bacterium. In some embodiments,the nucleic acid comprises a tolR gene from a bacterial species,subspecies, serovar, or strain that is the same as the bacterial speciesof the recombinant bacterium.

In some embodiments, the nucleic acid comprises a tolR gene that isoperably-linked to a regulatable promoter (e.g., a sugar-regulatablepromoter). In some embodiments, the nucleic acid comprises a tolR genethat is operably-linked to a sugar-regulatable promoter. In someembodiments, the sugar regulatable promoter exhibits increased activity(e.g., increased transcription) in the presence of a specific sugar anddecreased activity in the absence of a sugar. In some embodiments, thenucleic acid comprises a tolR gene that is operably-linked to arhamnose-regulatable promoter (e.g., a sugar-regulatable promoter). Insome embodiments, the nucleic acid comprises a tolR gene that isoperably-linked to an arabinose-regulatable promoter. In someembodiments, the arabinose-regulatable promoter is P_(BAD). In someembodiments, the recombinant bacterium comprises the mutationΔP_(tolR)::TT araC P_(araBAD) tolR.

7. Endosomal Escape Genes

In some embodiments, the recombinant bacterium has beengenetically-altered such that the bacterium is capable of escaping theendosomal compartment of a host cell. A recombinant bacterium mayexhibit a temporal delay in escaping an endosome following invasion ofthe host cell. Methods of detecting escape from an endosomal compartmentof a host cell are well known in the art, and include, for example,microscopic analysis.

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous sifA gene. In some embodiments, the recombinant bacteriumcomprises a mutation that alters the function of SifA. SifA is aneffector protein necessary for the formation of Salmonella-inducedfilaments and for the maintenance of the vacuolar membrane enclosing thebacterium. Bacteria comprising a deletion of sifA are capable ofescaping the host cell endosome (also called the Salmonella-containingvesicle, or SCV) following cellular invasion. In some embodiments, thedeletion of the endogenous sifA gene is a partial deletion. In someembodiments, the deletion of the endogenous sifA gene is a full-lengthdeletion. In some embodiments, the endogenous sifA gene is geneticallyaltered to insert a transcriptional terminator in the open reading frameof the gene. In some embodiments, a regulatory region of the endogenoussifA gene is genetically-modified to alter (e.g., decrease) theexpression of the sifA gene. In some embodiments, the promoter of anendogenous sifA gene is altered to include one or more regulatoryelements (e.g., a sugar-responsive promoter).

In some embodiments, the recombinant bacterium described herein ismodified to comprise a nucleic acid comprising a sifA gene. In someembodiments, the nucleic acid comprising a sifA gene is located on aplasmid in the bacterium. In some embodiments, the nucleic acidcomprising a sifA gene is located on a chromosome of the bacterium. Insome embodiments, the nucleic acid comprising a sifA gene is located atthe chromosomal locus corresponding to the locus of an endogenous a sifAthat has been deleted or altered in the bacterial chromosome. In someembodiments, the recombinant bacterium is modified to comprise a nucleicacid comprising a sifA gene, whereby an endogenous copy of the sifA genein the bacterial chromosome has been altered and/or deleted.

The nucleic acid sequence of an exemplary Salmonella sifA gene isprovided below:

(SEQ ID NO: 7) atgccgattactatagggaatggttttttaaaaagtgaaatccttaccaactccccaaggaatacgaaagaagcatggtggaaagttttatgggaaaaaattaaagacttctttttttctactggcaaagcaaaagcggaccgttgtctacatgagatgttgtttgccgaacgcgcccccacacgagagcggcttacagagattttttttgagttgaaagagttagcctgcgcatcgcaaagagatagatttcaggttcataatcctcatgaaaatgatgccaccattattcttcgcatcatggatcaaaacgaagagaacgaattgttacgtatcactcaaaataccgatacctttagctgtgaagtcatggggaatctttattttttaatgaaagatcgcccggatattttaaaatcgcatccacaaatgacggccatgattaagagaagatatagcgaaatcgtagactaccccctcccttcgacattatgtctcaatcctgctggcgcgccgatattatcggttccattagacaacatagaggggtatttatatactgaattgagaaaaggacatttagatgggtggaaagcgcaagaaaaggcaacctacctggcagcgaaaattcagtctgggattgaaaagacaacgcgcattttacaccatgcgaatatatccgaaagtactcagcaaaacgcatttttagaaacaatggcgatgtgtggattaaaacagcttgaaataccaccaccgcatacccacatacctattgaaaaaatggtaaaagaggttttactagcggataagacgtttcaggcgttcctcgtaacggatcccagcaccagccaaagtatgttagctgagatagtcgaagccatctctgatcaggtttttcacgccatttttagaatagacccccaggctatacaaaaaatggcggaagaacagttaaccacgctacacgttcgctcagaacaacaaagcggctgtttatgtt gttttttataa. 

The amino acid sequence of the SifA protein encoded by the nucleic acidof SEQ ID NO: 7 is provided below:

(SEQ ID NO: 8) MPITIGNGFLKSEILTNSPRNTKEAWWKVLWEKIKDFFFSTGKAKADRCLHEMLFAERAPTRERLTEIFFELKELACASQRDRFQVHNPHENDATIILRIMDQNEENELLRITQNTDTFSCEVIVIGNLYFLMKDRPDILKSHPQMTAMIKRRYSEIVDYPLPSTLCLNPAGAPILSVPLDNIEGYLYTELRKGHLDGWKAQEKATYLAAKIQSGIEKTTRILHHANISESTQQNAFLETMAMCGLKQLEIPPPHTHIPIEKMVKEVLLADKTFQAFLVTDPSTSQSMLAEIVEAISDQVFHAIFRIDPQAIQKMAEEQLTTLHVRSEQQSGCLCCFL. 

In some embodiments, the nucleic acid comprises a Salmonella sijA gene(provided as SEQ ID NO: 7). In some embodiments, the nucleic acidcomprises a sifA gene, wherein the sifA gene comprises a nucleic acidsequence that is at least 75%, at least 80%, at least 81%, at least 82%,at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the nucleic acid sequenceof SEQ ID NO: 7. In some embodiments, the nucleic acid comprises a sifAgene, wherein the sifA gene comprises a nucleic acid sequence that is atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% homologous to the nucleic acid sequence of SEQ ID NO:7.

In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding a SifA protein, wherein said SifA protein comprises an aminoacid sequence that is at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the amino acidsequence of SEQ ID NO: 8. In some embodiments, the nucleic acidcomprises a nucleic acid sequence encoding a SifA protein, wherein saidSifA protein comprises an amino acid sequence that is at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% homologous to the nucleic acid sequence of SEQ ID NO: 8.

In some embodiments, the nucleic acid comprises a sifA gene from abacterial species, subspecies, serovar, or strain that is different thanthe bacterial species of the recombinant bacterium. In some embodiments,the nucleic acid comprises a sifA gene from a bacterial species,subspecies, serovar, or strain that is the same as the bacterial speciesof the recombinant bacterium.

In some embodiments, the nucleic acid comprises a sifA gene that isoperably-linked to a regulatable promoter (e.g., a sugar-regulatablepromoter). In some embodiments, the nucleic acid comprises a sifA genethat is operably-linked to a sugar-regulatable promoter. In someembodiments, the sugar regulatable promoter exhibits increased activity(e.g., increased transcription) in the presence of a specific sugar anddecreased activity in the absence of a sugar. In some embodiments, thenucleic acid comprises a sifA gene that is operably-linked to arhamnose-regulatable promoter (e.g., a sugar-regulatable promoter). Insome embodiments, the nucleic acid comprises a sifA gene that isoperably-linked to an arabinose-regulatable promoter. In someembodiments, the arabinose-regulatable promoter is P_(BAD). In someembodiments, the recombinant bacterium comprises the mutation ΔsifA::TTaraC P_(BAD) sifA. In some embodiments, the recombinant bacteriumcomprises the mutation ΔP_(sifA)::TT araC P_(araBAD) sifA. When theexpression of the nucleic acid comprising a sifA gene is under thecontrol of an arabinose-regulated promoter, the bacterial escape fromthe host endosome can be delayed. Since arabinose is absent in hostcells, arabinose cannot induce the expression of the sifA gene. Thus, ifthe recombinant bacterium is cultured in the presence of arabinose priorto administration to the subject, the expression of sifA will graduallydecrease with each round of bacterial cell division thereby allowingescape of the bacterium from the host cell endosome during the initialcell division cycles. Similar delayed-escape mutations may beconstructed using other regulatable promoters, such as from thexylose-regulatable or rhamnose-regulatable promoter systems.

8. GTP Pyrophosphokinase Genes

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous relA gene, which encodes the GTP pyrophosphokinase RelA.The inclusion of a relA deletion in the recombinant bacterium uncouplesthe occurrence of growth-dependent lysis to the need for continuedprotein synthesis. In some embodiments, the deletion of the endogenousrelA gene is a partial deletion. In some embodiments, the deletion ofthe endogenous relA gene is a full-length deletion.

9. Other Attenuation Methods

Other methods of attenuation are known in the art. For instance,attenuation may be accomplished by altering (e.g., deleting) nativenucleic acid sequences found in the wild-type bacterium. For instance,if the bacterium is Salmonella, non-limiting examples of nucleic acidsequences which may be used for attenuation include: apab nucleic acidsequence, apur nucleic acid sequence, an aro nucleic acid sequence, asd,a dap nucleic acid sequence, nadA, pncB, galE, pmi, fur, rpsL, ompR,htrA, hemA, cdt, cya, crp, dam, phoP, phoQ, rfc, poxA, galU, mviA, sodC,recA, ssrA, sirA, inv, hilA, rpoE, flgM, tonB, slyA, and any combinationthereof. Exemplary attenuating mutations may be aroA, aroC, aroD, cdt,cya, crp, phoP, phoQ, ompR, galE, and htrA.

In certain embodiments, the above nucleic acid sequences may be placedunder the control of a sugar regulated promoter wherein the sugar ispresent during in vitro growth of the recombinant bacterium, butsubstantially absent within an animal or human host. The cessation intranscription of the nucleic acid sequences listed above would thenresult in attenuation and the inability of the recombinant bacterium toinduce disease symptoms.

B. Additional Mutations

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous recF gene, which encodes the DNA replication and repairprotein RecF. In some embodiments, the deletion of the endogenous recFgene is a partial deletion. In some embodiments, the deletion of theendogenous recF gene is a full-length deletion. In some embodiments, theendogenous recF gene is genetically altered to insert a transcriptionalterminator in the open reading frame of the gene.

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous recJ gene, which encodes the exonuclease RecJ. In someembodiments, the deletion of the endogenous recJ gene is a partialdeletion. In some embodiments, the deletion of the endogenous recJ geneis a full-length deletion. In some embodiments, the endogenous recJ geneis genetically altered to insert a transcriptional terminator in theopen reading frame of the gene.

The bacterium may also be modified to create a balanced-lethalhost-vector system, although other types of systems may also be used(e.g., creating complementation heterozygotes). For the balanced-lethalhost-vector system, the bacterium may be modified by manipulating itsability to synthesize various essential constituents needed forsynthesis of the rigid peptidoglycan layer of its cell wall. In oneexample, the constituent is diaminopimelic acid (DAP). Various enzymesare involved in the eventual synthesis of DAP.

In some embodiments, the recombinant bacterium comprises a deletion inan endogenous asd gene. In some embodiments, the deletion of theendogenous asd gene is a partial deletion. In some embodiments, thedeletion of the endogenous asd gene is a full-length deletion. In someembodiments, the endogenous asd gene is genetically altered to insert atranscriptional terminator in the open reading frame of the gene. Insome embodiments, the promoter of a endogenous asd gene is altered toinclude one or more regulatory elements (e.g., a sugar-responsivepromoter). In one example, the bacterium is modified by using a AasdAmutation to eliminate the bacterium's ability to produce β-aspartatesemialdehyde dehydrogenase, an enzyme essential for the synthesis ofDAP. Other mutations that result in the abolition of the synthesis ofDAP include, but are not limited to, dapA, dapB, dapC, dapD, dapE, dapF,and asd (see, e.g., U.S. Pat. No. 6,872,547, incorporated herein byreference). Other modifications that may be employed includemodifications to a bacterium's ability to synthesize D-alanine or tosynthesize D-glutamic acid (e.g., Amurl mutations), which are bothunique constituents of the peptidoglycan layer of the bacterial cellwall.

Similarly, various embodiments may comprise the araC P_(araBAD) c2 genecassette inserted into the asd nucleic acid sequence that encodesaspartate semialdehyde dehydrogenase. Since the araC nucleic acidsequence is transcribed in a direction that could lead to interferencein the expression of adjacent nucleic acid sequences and adverselyaffect vaccine strain performance, a transcription termination (TT)sequence is generally inserted 3′ to the araC nucleic acid sequence. Thechromosomal asd nucleic acid sequence is typically inactivated to enableuse of plasmid vectors encoding the wild-type asd nucleic acid sequencein the balanced lethal host-vector system. This allows for stablemaintenance of plasmids in vivo in the absence of any drug resistanceattributes that are not permissible in live bacterial vaccines. In someof these embodiments, the wild-type asd nucleic acid sequence may beencoded by the vector described herein. The vector enables the regulatedexpression of an antigen encoding sequence through the repressiblepromoter.

C. Repressor Regulatory Systems

In some embodiments, the recombinant bacterium comprises a nucleic acid(e.g., a gene) that is operably linked to a repressor-regulatablepromoter to facilitate the regulatable expression of the gene. Thus, insome embodiments, the recombinant bacterium comprises a nucleic acidcomprising a gene encoding a repressor. In some embodiments, the geneencoding the repressor is operably-linked to a regulatable promoter.Methods of chromosomally integrating a nucleic acid sequence encoding arepressor operably-linked to a regulatable promoter are known in the artand detailed in the examples. In some embodiments, the nucleic acidsequence encoding a repressor is not integrated into a chromosomal locussuch that the ability of the bacterium to colonize a host cell isdisrupted. In some embodiments, the recombinant bacterium comprises anucleic acid encoding a repressor that is integrated into the relA locusof the bacterial chromosome. In some embodiments, the recombinantbacterium comprises a nucleic acid encoding a repressor that isintegrated into the endA locus of the bacterial chromosome. In someembodiments, the recombinant bacterium comprises at least one nucleicacid sequence encoding a repressor. In some embodiments, the recombinantbacterium comprises at least two, at least three, at least four, atleast five, at least six or more nucleic acids encoding a repressor. Insome embodiments, the nucleic acid encoding the repressor is present ona plasmid in the bacterium. In some embodiments, the nucleic acidencoding the repressor is located in the bacterial chromosome. If thereis more than one nucleic acid sequence encoding a repressor, eachnucleic acid sequence encoding a repressor may be operably linked to aregulatable promoter, such that each promoter is regulated by the samecompound or condition. Alternatively, each nucleic acid sequenceencoding a repressor may be operably linked to a regulatable promoter,each of which is regulated by a different compound or condition.

As used herein, a “repressor” refers to a biomolecule that represses thetranscriptional activity of a promoter. In some embodiments, therepressor is synthesized by the recombinant bacterium in high enoughquantities during in vitro culture, such that the transcription of anucleic acid that is operably linked to a repressor-regulatable promoteris repressed. This may be particularly advantageous if, for example,expression of the product encoded by said nucleic acid impedes the invitro growth of the bacterium, and/or the ability of the bacterium toinfect and/or colonize a subject. In some embodiments, the nucleic acidthat is operably-linked to the repressor-regulatable promoter expressesan antigen of interest. In some embodiments, the concentration of therepressor within the cell gradually decreases with each cell divisioncycle after transcription of the gene encoding the repressor decreasesor ceases (e.g., in vivo). The use of a particular repressor, asdescribed herein, may depend, in part, on the species, subspecies,strain or serovar of the recombinant bacterium being used. In someembodiments, the repressor is derived from the same species (e.g., thesame bacterial species or the same phage) from which therepressor-regulatable promoter is derived. In some embodiments therepressor is not derived from the same bacterial species as thebacterial species in which the repressor is expressed. For example, insome embodiments, the repressor is derived from E. coli if therecombinant bacterium is of the genus Salmonella. Other suitablerepressors include repressors derived from a bacteriophage.

A nucleic acid sequence encoding a repressor and regulatable promoterdetailed above may be modified so as to optimize the expression level ofthe nucleic acid sequence encoding the repressor. The optimal level ofexpression of the nucleic acid sequence encoding the repressor may beestimated, or may be determined by experimentation. Such a determinationshould take into consideration whether the repressor acts as a monomer,dimer, trimer, tetramer, or higher multiple, and should also take intoconsideration the copy number of the vector encoding the antigen ofinterest. In an exemplary embodiment, the level of expression isoptimized so that the repressor is synthesized while in a permissiveenvironment (i.e., in vitro growth) at a level that substantiallyinhibits the expression of the nucleic acid encoding an antigen ofinterest, and is substantially not synthesized in a non-permissiveenvironment, thereby allowing expression of the nucleic acid encoding anantigen of interest.

In some embodiments, the recombinant bacterium described herein ismodified to comprise a nucleic acid comprising a lacI gene, whichencodes the LacI repressor protein. The expression of the lacI-encodedrepressor in the recombinant bacterium described herein may be used toregulate the expression of a gene encoding an antigen of interestexpressed by the bacterium. For example, in some embodiments, theexpression of the lacI gene is regulated by a sugar-regulatable promoter(e.g., an arabinose-regulatable promoter). When cultured in the presenceof arabinose, the recombinant bacterium will synthesize the LacIrepressor protein, which in turn will repress the expression of a geneencoding an antigen of interest that is operably-linked to aLacI-responsive promoter (e.g., P_(trc), P_(lac), P_(T7lac) andP_(tac)). Upon administration to the subject and in the absence of asource of arabinose, the synthesis of LacI repressor ceases, leading tode-repression of the LacI-responsive promoter and the subsequencecausing expression of the antigen of interest. The concentration of LacIin the cell decreases by about half at each cell division in vivo,leading to a gradual decreased level of repression and gradual increasedsynthesis of the antigen of interest.

In some embodiments, the nucleic acid comprising a lacI gene is locatedon a plasmid in the bacterium. In some embodiments, the nucleic acidcomprising a lacI gene is located on a chromosome of the bacterium. Insome embodiments, the nucleic acid comprising a lacI gene is located atthe chromosomal locus corresponding to the locus of an endogenous -relAgene that has been deleted or altered in the bacterial chromosome. Insome embodiments, the recombinant bacterium is modified to comprise anucleic acid comprising a lacI gene, whereby an endogenous copy of thelacI gene in the bacterial chromosome has been altered and/or deleted.

In some embodiments, the nucleic acid comprises an Escherichia coli lacIgene. The nucleic acid sequence of the E. coli lacI gene is providedbelow:

(SEQ ID NO: 9) gtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatageggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtga. 

The amino acid sequence of the E. coli LacI protein encoded by thenucleic acid of SEQ ID NO: 9 is provided below:

(SEQ ID NO: 10) MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLA RQVSRLESGQ. 

In some embodiments, the nucleic acid comprises a lacI gene, wherein thelacI gene comprises a nucleic acid sequence that is at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the nucleic acid sequence of SEQ ID NO: 9. In someembodiments, the nucleic acid comprises a lacI gene, wherein the lacIgene comprises a nucleic acid sequence that is at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%homologous to the nucleic acid sequence of SEQ ID NO: 9.

In some embodiments, the nucleic acid comprises a nucleic acid sequenceencoding a LacI protein, wherein said LacI protein comprises an aminoacid sequence that is at least 75%, at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the amino acidsequence of SEQ ID NO: 10. In some embodiments, the nucleic acidcomprises a nucleic acid sequence encoding a LacI protein, wherein saidLacI protein comprises an amino acid sequence that is at least 75%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% homologous to the nucleic acid sequence of SEQ ID NO: 10.

In some embodiments, the nucleic acid comprises a lacI gene that isoperably-linked to a regulatable promoter (e.g., a sugar-regulatablepromoter). In some embodiments, the nucleic acid comprises a lacI genethat is operably-linked to a sugar-regulatable promoter. In someembodiments, the sugar regulatable promoter exhibits increased activity(e.g., increased transcription) in the presence of a specific sugar anddecreased activity in the absence of a sugar. In some embodiments, thenucleic acid comprises a lacI gene that is operably-linked to arhamnose-regulatable promoter (e.g., a sugar-regulatable promoter). Insome embodiments, the nucleic acid comprises a lacI gene that isoperably-linked to an arabinose-regulatable promoter. In someembodiments, the arabinose-regulatable promoter is P_(araBAD). In someembodiments, the recombinant bacterium comprises the mutationΔrelA::araC P_(araBAD) lacI TT.

D. Antigens

In some embodiment, the recombinant bacterium comprises a nucleic acidencoding an antigen of interest. As used herein, “antigen” refers to abiomolecule capable of eliciting an immune response in a host. In someembodiments, an antigen may be a protein, or fragment of a protein. Insome embodiments, the recombinant bacterium comprises a nucleic acid(e.g., a plasmid) encoding an antigen of interest, wherein the nucleicacid is expressed by the host cell (e.g., a DNA vaccine). In anexemplary embodiment, the antigen elicits a protective immune responsein a subject.

As used herein, “protective” means that the immune response contributesto the lessening of any symptoms associated with infection of a hostwith the pathogen the antigen was derived from or designed to elicit aresponse against. For example, a protective antigen from a pathogen,such as Salmonella, may induce an immune response that helps toameliorate symptoms associated with Salmonella infection or reduce themorbidity and mortality associated with infection with the pathogen ormay reduce the ability of Salmonella to infect and colonize the host.The use of the term “protective” in this disclosure does not necessarilyrequire that the host is completely protected from the effects of thepathogen.

In some embodiments, the antigen of interest is an antigen derived froman infectious agent. In some embodiments, the antigen of interest isderived from an infectious agent selected from the group consisting of avirus, a bacterium, a protozoan, a prion, a fungus, and a helminth. Insome embodiments, the antigen of interest is derived from a bacterium.In some embodiments, the antigen of interest is a Salmonella antigen. Insome embodiments, the Salmonella antigen is selected from the groupFliC, FliC180, OmpC, OmpD, OmpF, SseB, and SseI. In some embodiments,the antigen of interest is a viral antigen. In some embodiments, theantigen of interest is an influenza antigen. In some embodiments, theinfluenza antigen is hemagglutinin or neuraminidase, if delivered by aDNA vaccine. In some embodiments, the antigen of interest is an antigenassociated with cancer. In some embodiments, the antigen associated withcancer is selected from the group consisting of MAGE-A, MAGE-C1, BAGE,GAGE, XAGE, NY-ESO1 (also known as CTAG1B and LAGE2), LAGE1 (also knownas CTAG2) and survivin.

Alternatively, antigens may be derived from gametes, provided they aregamete specific, and may be designed to block fertilization. In anotheralternative, antigens may be tumor antigens, and may be designed todecrease tumor growth. It is specifically contemplated that antigensfrom organisms newly identified or newly associated with a disease orpathogenic condition, or new or emerging pathogens of animals or humans,including those now known or identified in the future, may be expressedby a bacterium detailed herein. Furthermore, antigens are not limited tothose from pathogenic organisms.

Immunogenicity of the bacterium may be augmented and/or modulated byconstructing strains that also express sequences for cytokines,adjuvants, and other immunomodulators.

Some examples of microorganisms useful as a source for antigen arelisted below. These may include microorganisms for the control of plaguecaused by Yersinia pestis and other Yersinia species such as Y.pseudotuberculosis and Y. enterocolitica, for the control of gonorrheacaused by Neisseria gonorrhoea, for the control of syphilis caused byTreponema pallidum, and for the control of venereal diseases as well aseye infections caused by Chlamydia trachomatis. Species of Streptococcusfrom both group A and group B, such as those species that cause sorethroat or heart diseases, Streptococcus equi, which causes strangles inequines, Streptococcus mutans, which causes cavities, and Streptococcuspneumoniae, Erysipelothrix rhusiopathiae, Neisseria meningitidis,Mycoplasma pneumoniae and other Mycoplasma-species, Hemophilusinfluenza, Bordetella pertussis, Mycobacterium tuberculosis,Mycobacterium leprae, other Bordetella species, Escherichia coli,Brucella abortus, Pasteurella hemolytica and P. multocida, Vibriocholera, Shigella species, Borrellia species, Bartonella species,Heliobacter pylori, Campylobacter species, Pseudomonas species,Moraxella species, Brucella species, Francisella species, Aeromonasspecies, Actinobacillus species, Clostridium species (such as C.perfringens), Rickettsia species, Bacillus species, Coxiella species,Ehrlichia species, Listeria species, and Legionella pneumophila areadditional examples of bacteria within the scope of this disclosure fromwhich antigen nucleic acid sequences could be obtained.

Viral antigens may also be used. Viral antigens may be used in antigendelivery microorganisms directed against viruses, either DNA or RNAviruses, for example from the classes Papovavirus, Adenovirus,Herpesvirus, Poxvirus, Parvovirus, Reovirus, Picornavirus, Myxovirus,Paramyxovirus, Flavivirus or Retrovirus. Antigens may also be derivedfrom pathogenic fungi, protozoa and parasites. However, means of antigendelivery or sequences encoding the antigen depends on the type ofantigen and/or virus.

In certain embodiments, an antigen may comprise a B cell epitope or a Tcell epitope. Alternatively, an antigen to which an immune response isdesired may be expressed as a fusion to a carrier protein that containsa strong promiscuous T cell epitope and/or serves as an adjuvant and/orfacilitates presentation of the antigen to enhance, in all cases, theimmune response to the antigen or its component part. This can beaccomplished by methods known in the art. Fusion to tenus toxin fragmentC, CT-B, LT-B and hepatitis virus B core are particularly useful forthese purposes, although other epitope presentation systems are wellknown in the art.

In further embodiments, a nucleic acid sequence encoding an antigen maycomprise a secretion signal.

As stated above, the level of synthesis of an antigen of interest may beoptimized by modifying the nucleic acid sequence encoding the repressorand/or promoter. As used herein, “modify” refers to an alteration of thenucleic acid sequence of the repressor and/or promoter that results in achange in the level of transcription of the nucleic acid sequenceencoding the repressor, or that results in a change in the level ofsynthesis of the repressor. For instance, in one embodiment, modify mayrefer to altering the start codon of the nucleic acid sequence encodingthe repressor. Generally speaking, a GTG or TTG start codon, as opposedto an ATG start codon, may decrease translation efficiency ten-fold. Inanother embodiment, modify may refer to altering the Shine-Dalgarno (SD)sequence of the nucleic acid sequence encoding the repressor. The SDsequence is a ribosomal binding site generally located 6-7 nucleotidesupstream of the start codon. The SD consensus sequence is AGGAGG, andvariations of the consensus sequence may alter translation efficiency.In yet another embodiment, modify may refer to altering the distancebetween the SD sequence and the start codon. In still anotherembodiment, modify may refer to altering the −35 sequence for RNApolymerase recognition. In a similar embodiment, modify may refer toaltering the −10 sequence for RNA polymerase binding. In an additionalembodiment, modify may refer to altering the number of nucleotidesbetween the −35 and −10 sequences. In an alternative embodiment, modifymay refer to optimizing the codons of the nucleic acid sequence encodingthe repressor to alter the level of translation of the mRNA encoding therepressor. For instance, non-A rich codons initially after the startcodon of the nucleic acid sequence encoding the repressor may notmaximize translation of the mRNA encoding the repressor. Similarly, thecodons of the nucleic acid sequence encoding any of the proteinsdescribed herein may be codon-optimized, i.e., altered so as to mimicthe codons from highly synthesized proteins of a particular organism. Ina further embodiment, modify may refer to altering the GC content of thenucleic acid sequence encoding the repressor to change the level oftranslation of the mRNA encoding the repressor. Methods of modifying anucleic acid sequence are known in the art.

In some embodiments, more than one modification or type of modificationmay be performed to optimize the expression level of a nucleic aciddescribed herein (e.g., a nucleic acid encoding a repressor or antigenof interest). For instance, at least one, two, three, four, five, six,seven, eight, or nine modifications, or types of modifications, may beperformed to optimize the expression level of a nucleic acid describedherein. By way of non-limiting example, when the repressor is LacI, thenthe nucleic acid sequence of LacI and the promoter may be altered so asto increase the level of LacI synthesis. In one embodiment, the startcodon of the LacI repressor may be altered from GTG to ATG. In anotherembodiment, the SD sequence may be altered from AGGG to AGGA. In yetanother embodiment, the codons of lacI may be optimized according to thecodon usage for highly synthesized proteins of Salmonella. In a furtherembodiment, the start codon of lacI may be altered, the SD sequence maybe altered, and the codons of lacI may be optimized.

In some embodiments, the recombinant bacterium comprises a nucleic acidthat is located in a plasmid or vector. As used herein, “vector” refersto an autonomously replicating nucleic acid unit. The present disclosurecan be practiced with any known type of vector, including viral, cosmid,phasmid, and plasmid vectors. The most preferred type of vector is aplasmid vector. In some embodiments, the plasmid or vector is a highcopy plasmid. In some embodiments, the plasmid or vector is a low copyplasmid or vector.

As is well known in the art, plasmids and other vectors may possess awide array of promoters, multiple cloning sequences, transcriptionterminators, etc., and vectors may be selected so as to control thelevel of expression of the nucleic acid sequence encoding an antigen bycontrolling the relative copy number of the vector. In some instances inwhich the vector might encode a surface localized adhesin as theantigen, or an antigen capable of stimulating T-cell immunity, it may bepreferable to use a vector with a low copy number such as at least two,three, four, five, six, seven, eight, nine, or ten copies per bacterialcell. A non-limiting example of a low copy number vector may be a vectorcomprising the pSC101 ori.

In some embodiments, the plasmid comprises a nucleic acid sequenceencoding an aspartate-semialdehyde dehydrogenase gene (e.g., asdA).These plasmids may be advantageously used to complement a bacterium thatcomprises an aspartate-semialdehyde dehydrogenase gene mutation (e.g.,asdA). In some embodiments, the plasmid is selected from the groupconsisting of pYA3342, pYA3337, and pYA3332.

In other cases, an intermediate copy number vector might be optimal forinducing desired immune responses. For instance, an intermediate copynumber vector may have at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 copies per bacterial cell.A non-limiting example of an intermediate copy number vector may be avector comprising the p15A ori.

In still other cases, a high copy number vector might be optimal for theinduction of maximal antibody responses. A high copy number vector mayhave at least 31, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100 copies per bacterial cell. In some embodiments, a high copy numbervector may have at least 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, or 400 copies per bacterial cell. Non-limiting examplesof high copy number vectors may include a vector comprising the pBR orior the pUC ori.

Additionally, vector copy number may be increased by selecting formutations that increase plasmid copy number. These mutations may occurin the bacterial chromosome but are more likely to occur in the plasmidvector.

Preferably, vectors used herein do not comprise antibiotic resistancemarkers to select for maintenance of the vector.

Promoters for use in the embodiments described herein are known in theart. One of skill in the art would recognize that the selection of arepressor dictates, in part, the selection of the promoter to be used toregulate the expression of a nucleic acid described herein. Forinstance, if the repressor is LacI, then the promoter may be selectedfrom the group consisting of LacI responsive promoters, such as P_(trc),P_(lac), P_(T7lac), P_(tac), P_(ompA lacO), and P_(lpp lacO). If therepressor is C2, then the promoter may be selected from the groupconsisting of C2 responsive promoters, such as P22 promoters P_(L) andP_(R). If the repressor is C1, then the promoter may be selected fromthe group consisting of C1 responsive promoters, such as X promotersP_(L) and P_(R).

In each embodiment herein, the promoter regulates expression of anucleic acid sequence. In some embodiments, the promoter comprises aregulatory sequence controlled by a repressor, such that expression ofthe nucleic acid sequence is repressed when the repressor is synthesized(e.g., during in vitro growth of the bacterium), but expression of thenucleic acid sequence encoding an antigen is high when the repressor isnot synthesized (e.g., in vivo). Generally speaking, the concentrationof the repressor will decrease with every cell division after expressionof the gene encoding the repressor ceases. In some embodiments, theconcentration of the repressor decreases such that high levels ofexpression of the nucleic acid sequence that is being regulated isachieved after about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 divisions ofthe bacterium. In an exemplary embodiment, the concentration of therepressor decreases enough to allow high-level expression of the nucleicacid sequence encoding an antigen after about 5 divisions of thebacterium in vivo.

In certain embodiments, the promoter may comprise other regulatoryelements. For instance, the promoter may comprise lacO if the repressoris LacI. This is the case with the lipoprotein promoter P_(lpp lacO)that is regulated by LacI since it possesses the LacI binding domainlacO. In one embodiment, the repressor is a LacI repressor and thepromoter is P_(trc).

In some embodiments, the expression of the nucleic acid sequenceregulated by a repressor is repressed in vivo. Expression may be“repressed” or “partially repressed” when it is about 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or even less than 1% of theexpression under non-repressed conditions. Thus although the level ofexpression under conditions of “complete repression” might be exceedinglow, it is likely to be detectable using very sensitive methods sincerepression can never by absolute.

Conversely, the expression of the nucleic acid sequence encoding theantigen should be high when the expression of the repressor isrepressed. For instance, if the repressor is not synthesized duringgrowth of the recombinant bacterium in a host, the expression of thenucleic acid under the control of the repressor will be high. As usedherein, “high level” expression refers to expression that is strongenough to elicit an immune response to the antigen. Consequently, thecopy number correlating with high level expression can and will varydepending on the antigen and the type of immune response desired.Methods of determining whether an antigen elicits an immune responsesuch as by measuring antibody levels or antigen-dependent T cellpopulations or antigen-dependent cytokine levels are known in the art,and methods of measuring levels of expression of antigen encodingsequences by measuring levels of mRNA transcribed or by quantitating theexpression level of a protein are also known in the art.

In each of the above embodiments, a recombinant bacterium capable ofregulated expression may also be attenuated. “Attenuated” refers to thestate of the bacterium wherein the bacterium has been weakened from itswild-type fitness by some form of recombinant or physical manipulation.This includes altering the genotype of the bacterium to reduce itsability to cause disease. However, the bacterium's ability to colonizethe gut (in the case of Salmonella) and induce immune responses is,preferably, not substantially compromised.

In an exemplary embodiment, a recombinant bacterium may be attenuated asdescribed above. In which case, both regulated attenuation and regulatedexpression of an antigen encoding sequence may be dependent upon a sugarregulatable system. Consequently, the concentration of sugar (e.g.,arabinose) needed for optimal expression of the regulated antigenencoding sequence may not be the same as the concentration for optimalexpression of attenuation. In an exemplary embodiment, the concentrationof arabinose for the optimization of both regulated attenuation andregulated expression of sequences encoding antigen will be substantiallythe same.

Accordingly, the promoter and/or the nucleic acid sequence encoding anattenuation protein may be modified to optimize the system. Methods ofmodification are detailed above. Briefly, for example, the SD ribosomebinding sequence may be altered, and/or the start codon may be alteredfrom ATG to GTG for the nucleic acid sequences fur and phoPQ, so thatthe production levels of Fur and PhoPQ are optimal for both theregulated attenuation phenotype and the regulated expression whengrowing strains with a given concentration of arabinose. One of skill inthe art will appreciate that other nucleic acid sequences, in additionto fur and phoPQ, may also be altered as described herein in combinationwith other well-known protocols. In addition, these attenuating nucleicacid sequences may be regulated by other systems using well-establishedprotocols known to one of skill in the art. For example, they may beregulated using with promoters dependent on addition of maltose,rhamnose, or xylose rather than arabinose.

II. Pharmaceutical Compositions

A recombinant bacterium may be administered to a host as apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition may be used as a vaccine to elicit an immune response to therecombinant bacterium, including any antigens that may be synthesizedand delivered by the bacterium. In an exemplary embodiment, the immuneresponse is protective. Immune responses to antigens are well studiedand widely reported.

Pharmaceutical compositions may be administered to any host capable ofmounting an immune response. Such hosts may include all vertebrates, forexample, mammals, including domestic animals, agricultural animals,laboratory animals, and humans, and various species of birds, includingdomestic birds and birds of agricultural importance. Preferably, thehost is a warm-blooded animal. In one embodiment, the host is a cow. Insome embodiments, the host is an equine. In another embodiment, the hostis an avian. In another embodiment, the host is a human. Thepharmaceutical composition can be administered to the subject as aprophylactic or for treatment purposes.

In some embodiments, the recombinant bacterium is alive whenadministered to a host in a pharmaceutical composition described herein.Suitable vaccine composition formulations and methods of administrationare detailed below.

A pharmaceutical composition comprising a recombinant bacterium mayoptionally comprise one or more possible additives, such as carriers,preservatives, stabilizers, adjuvants, and other substances.

In one embodiment, the pharmaceutical composition comprises an adjuvant.Adjuvants are optionally added to increase the ability of the vaccine totrigger, enhance, or prolong an immune response. In exemplaryembodiments, the use of a live attenuated recombinant bacterium may actas a natural adjuvant. In some embodiments, the recombinant bacteriumsynthesizes and secretes an immune modulator. Additional materials, suchas cytokines, chemokines, and bacterial nucleic acid sequences naturallyfound in bacteria, like CpG, are also potential vaccine adjuvants.

In some embodiments, the pharmaceutical composition comprises bufferedsaline (e.g., phosphate-buffered saline (PBS)).

In some embodiments, the pharmaceutical composition comprises a foodproduct.

In another embodiment, the pharmaceutical may comprise a pharmaceuticalcarrier (or excipient). Such a carrier may be any solvent or solidmaterial for encapsulation that is non-toxic to the inoculated host andcompatible with the recombinant bacterium. A carrier may give form orconsistency, or act as a diluent. Suitable pharmaceutical carriers mayinclude liquid carriers, such as normal saline and other non-toxic saltsat or near physiological concentrations, and solid carriers not used forhumans, such as talc or sucrose, or animal feed. Carriers may alsoinclude stabilizing agents, wetting and emulsifying agents, salts forvarying osmolarity, encapsulating agents, buffers, and skin penetrationenhancers. Carriers and excipients as well as formulations forparenteral and nonparenteral drug delivery are set forth in Remington'sPharmaceutical Sciences 19th Ed. Mack Publishing (1995). When used foradministering via the bronchial tubes, the pharmaceutical composition ispreferably presented in the form of an aerosol.

In some embodiments, the pharmaceutical composition is delivered to afarm animal (e.g., poultry). In some embodiments, the pharmaceuticalcomposition is delivered as a course spray (e.g., for use in hatcheriesfor delivery to poultry). In some embodiments, the pharmaceuticalcomposition is delivered in the drinking water.

Care should be taken when using additives so that the live recombinantbacterium is not killed, or have its ability to effectively colonizelymphoid tissues such as the GALT, NALT and BALT compromised by the useof additives. Stabilizers, such as lactose or monosodium glutamate(MSG), may be added to stabilize the pharmaceutical composition againsta variety of conditions, such as temperature variations or afreeze-drying process. The recombinant bacterium may also beco-administered with glutamate and/or arginine as described herein.

The dosages of a pharmaceutical composition can and will vary dependingon the recombinant bacterium, the regulated antigen, and the intendedhost, as will be appreciated by one of skill in the art. Generallyspeaking, the dosage need only be sufficient to elicit a protectiveimmune response in a majority of hosts. Routine experimentation mayreadily establish the required dosage. Typical initial dosages ofvaccine for oral administration could be about 1×10⁷ to 1×10¹⁰ CFUdepending upon the age of the host to be immunized. Administeringmultiple dosages may also be used as needed to provide the desired levelof protective immunity.

In order to stimulate a preferred response of the GALT, NALT or BALTcells, administration of the pharmaceutical composition directly intothe gut, nasopharynx, or bronchus is preferred, such as by oraladministration, intranasal administration, gastric intubation or in theform of aerosols, although other methods of administering therecombinant bacterium, such as intravenous, intramuscular, subcutaneousinjection or intramammary, intrapenial, intrarectal, vaginaladministration, or other parenteral routes, are possible, e.g., foranti-cancer applications.

In some embodiments, these compositions are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intradermally, intramuscularly, etc.).

In another embodiment, the disclosure provides a method for eliciting animmune response against an antigen in a host. The method comprisesadministering to the host an effective amount of a pharmaceuticalcomposition comprising a recombinant bacterium described herein.

In still another embodiment, a recombinant bacterium may be used in amethod for eliciting an immune response against a pathogen in anindividual in need thereof. The method comprises administrating to thehost an effective amount of a pharmaceutical composition comprising arecombinant bacterium as described herein. In a further embodiment, arecombinant bacterium described herein may be used in a method forameliorating one or more symptoms of an infectious disease in a host inneed thereof. The method comprises administering an effective amount ofa pharmaceutical composition comprising a recombinant bacterium asdescribed herein.

EXAMPLES

The present invention is further illustrated by the following examplesthat should not be construed as limiting in any way. The contents of allcited references, including literature references, issued patents, andpublished patent applications, as cited throughout this application arehereby expressly incorporated herein by reference. It should further beunderstood that the contents of all the figures and tables attachedhereto are also expressly incorporated herein by reference.

Example 1: Background

Protective immunity to Salmonella depends on the combined action ofspecific antibodies, B cells and T-cell-acquired immune responses(52-56). Effective clearance of primary infection requires a Th1response, with the help of antibody limiting bacteremia (55, 57).Antibody responses are important to achieve protection againstSalmonella infection (58-60), as seen for protection against S. Typhi inmice (61) and humans (62-64). RASVs induce all three branches of theimmune system (i.e., mucosal antibody and cellular responses, andsystemic antibody and cellular responses). All three of branches of theimmune system are important in conferring protective immunity toSalmonella and all pathogens that colonize on or invade through themucosal surface.

Salmonella possess a number of immunologically-related cross-reactiveantigens. These include the LPS core polysaccharide that is the same inmost, if not all, S. enterica serovars (65, 66) except for S. Arizonae(67, 68). In addition, OMPs, although possessing micro-heterogeneity,nevertheless share antigenic determinants (69), as well asiron-regulated outer membrane proteins (IROMPs) (70) that are requiredfor iron acquisition (70), an essential important function for pathogensuccess within an infected animal.

Salmonella vaccines can be used to display wild-type surface antigenicdeterminants in vitro and during the initial phase of infection throughmucosal surfaces in the orally immunized host and then cease tosynthesize LPS O-antigen side chains by a Δpmi mutation (71-74) and toconstitutively synthesize IROMPs in internal organs (75, 76) by aΔP_(fur)::TT araC P_(araBAD) fur (ΔP_(fur)) deletion-insertion mutation(77). S. Typhimurium strains with the Δpmi mutation are not completelyattenuated, but have high immunogenicity, efficacy in enhancinginduction of high antibody titers to cross-protective OMPs, IROMPs (76)and conserved LPS core (78, 79). However, the LPS core is not fullyexposed because there are still two sugars attached to the LPS core.Strains with the Δpmi mutation also enhance the production of OuterMembrane Vesicles (OMVs) that can deliver recombinant protectiveantigens for enhanced protective immunity (80). The ΔP_(fur) mutationenables expression of the fur gene to be solely dependent on thepresence of arabinose (75, 81, 82) and is blind to the concentration ofiron to achieve in vivo a high constitutive synthesis level of allcomponents for iron acquisition including immunologically cross-reactiveIROMPs. Immune responses to highly immunogenic IROMPs are effective inpreventing septicemic infection with enteropathogens (83). Antibodiesinduced to IROMPs from one bacterial serotype can recognize IROMPssynthesized by other serotypes (84). Two inactivated vaccines based onIROMP overproduction are licensed to protect against salmonellosis inpoultry (85, 86).

Live Salmonella delivering both surface polysaccharides and OMPs to theimmune system are more immunogenic than glycoconjugate vaccines.Salmonella spontaneously releases 50 to 90 nm bleb-like particles ofouter cell wall membrane (87-89). These blebs, called GMMA (GeneralizedModules for Membrane Antigens) or outer membrane vesicles, constitute anenriched source of outer membrane-associated antigens in their nativeconformation and correct orientation. GMMA or outer membrane vesiclesprovide significant advantages over recombinant proteins because theycontain multiple pathogen-associated molecular patterns (PAMPs),including TLR ligands, which have the potential to act as self-adjuvantsin the immune responses they elicit (90-95). GMMA or outer membranevesicles are also different from detergent extracted OMPs which lose anumber of outer membrane components, like lipoproteins, and thus resultin reduced immunogenicity. GMMA or outer membrane vesicles are currentlybeing explored as vaccines for meningococcus (96, 97), Shigella (87) andSalmonella (27). Preclinical studies with candidate GMMA or outermembrane vesicles vaccines indicate good immunogenicity and broad crossprotective immunity against a variety of strains (98). A prototypemeningococcal GMMA or outer membrane vesicles has been tested in onePhase 1 clinical trial without adverse effects (99) and a prototypeShigella GMMA or outer membrane vesicles is planned for a Phase 2 trial.GMMA or outer membrane vesicles production can be enhanced by deletionof the tolR gene (87, 100, 101), as seen in tolR mutants of Salmonellaand Shigella (87, 102). Deletions of genes, such as htrB (88) and msbB(103) for lipid A modification, can reduce reactogenicity. Although thenew GMMA or outer membrane vesicles vaccines have a reduced number ofpurification steps because they are spontaneously released byappropriate vaccine bacterial seed strains, downstream procedures, likecomplex tangential flow filtration, for GMMA or outer membrane vesiclespurification are still needed (87, 104, 105). In contrast, the instantdisclosure provides an in vivo GMMA or outer membrane vesiclesproduction system to omit downstream purification procedures withoutcompromising the efficiency.

Among surface-exposed or secreted protective antigens in Salmonella, 6antigens, FliC, OmpC, OmpD, OmpF, SseB, and SseI, may be used (106-113).These antigens are not the most abundant proteins in Salmonella (106).FliC synthesis is even deregulated at systemic sites (114-116).Preclinical studies in mice have demonstrated that immunization withthose above antigens could protect against Salmonella challenge (57,111, 117-120). OmpC and F induce long-lasting antibody responses in mice(121) and have been found to be safe and immunogenic when tested in aPhase 1 study in humans (122). OmpD is a key target for a protective Blbcell antibody response independent of T cells (57, 111) and is conservedin all serovars of Salmonella except serovar Typhi (123, 124). The SPI-2translocon subunit SseB plays the critical function for the secretion ofT3SS effector and replication of Salmonella in the cell (125). It is aserodominant target of adaptive immunity in children with Salmonellabacteremia (120) and encompasses multiple epitopes for CD4 T-cellimmunity in human volunteers (108, 120, 126). Another SPI-2 effector,SseI, plays a role in modulating the migration of infected cells, and isrequired for long-term systemic infection (127-131). Preserving thecorrect conformation of such antigens is critical as revealed by thefailure of recombinant Salmonella porins to protect mice (132). RASVenables delivery of these antigens in their correct conformation andorientation with high levels of production, combined with theself-adjuvanting properties of Salmonella that deliver innate signalsthrough TLR ligands and other PAMPs to induce Salmonella-specific T-celland B-cell immunity.

Disclosed herein is an innovative RASV platform to overproduceprotective Salmonella antigens in vivo. This system is a unique triplesugar regulated system, double shutoff of O-antigen synthesis byrhamnose and mannose and overproduction of GMMA or outer membranevesicles by arabinose. It will also incorporate the RDA and RDPSsystems. These systems will not increase the virulence (by theintroduction of these self-antigens) because most of the antigen genesare not highly expressed in vivo (106). The overexpression of antigengenes will also attenuate the strain (133, 134), as shown byoverexpression of the flagellin gene (133, 135). The virulence ofstrains with or without chromosomal mutations for these antigen geneswhen carrying antigen gene expression plasmids can also be evaluated, asdiscussed further below. In case the expression plasmid increases thevirulence high enough to cause disease, the strain or the plasmid can bemodified to guarantee the attenuation attributes. Levels of geneexpression can be modified up or down, as necessary, by switching thesugar regulated promoters, altering promoter and Shine-Dalgarnonucleotide sequence and the spacing between these elements and the startcodon of the regulated gene.

Example 2: Materials and Methods

Bacterial Strains, Media and Bacterial Growth:

Strain construction is performed in virulent S. Typhimurium strain χ3761(75) and S. Enteritidis χ3550 (136). Different virulent wild-typeSalmonella serovars, including S. Typhimurium χ3761 (B), S. Enteritidisχ3550 (D), S. Heidelberg χ3749 (B) (137), S. Choleraesuis χ3246 (C1)(138), S. Infantis χ3213 (C1) (139), S. Newport χ3240 (C2) (139), S.Dublin χ12323 (D) (140), are used for challenges. The LD₅₀s of most ofthese strains are between 10³ and 10⁵ in mice and chickens except thatS. Heidelberg, S. Infantis and S. Newport do not often cause lethaldisease in either mice or chickens. LB media or plates with appropriatesupplements when needed are used for growth of Salmonella (141, 142).

Molecular and Genetic Procedures.

Methods for DNA manipulations and PCR are standard (143). DNA sequenceanalysis is performed at the UFL DNA Sequence Laboratory whileoligonucleotide and/or gene segment syntheses will be obtainedcommercially. Construction of deletions or deletion/insertions inSalmonella is performed using suicide vectors or P22 transduction(144-146).

Strain Characterization.

Vaccine strains are fully characterized at each step in theirconstruction and before immunization studies for the presence of allphenotypes and genotypes. Genetic attributes are confirmed by PCR withappropriate probes and/or phenotype analyses. The fluorescent dye influxmethod is used to evaluate mutant membrane permeability. Strains arecompared with vector control strains for stability of plasmidmaintenance and antigen synthesis when strains are grown in the presenceof arabinose or other sugars and/or DAP over a 50 generation period(147). LPS is checked by silver staining (148). Growth curves will bedetermined for each strain. Other experiments include determining OMP(147) and IROMPs profiles (149), OMV (80) and GMMA characterization (87,150, 151), serum (152), bile and microbial peptides resistance (136),and attachment/invasion to epithelial INT-407 cells (153, 154). Eachstrain with antigen-specifying plasmid is evaluated for synthesis of theheterologous antigen by western blot.

Antigen Preparation.

Protective antigens, FliC, OmpC, OmpD, OmpF, SseB, and SseI, withC-terminal His-tag, are cloned into pBAD-His or pET vectors forsynthesis in E. coli Top 10 or BL21 and isolated by nickelchromatography (Sigma). Purified proteins are used for ELISA and ELISPOTassays and for preparing antiserum in New Zealand female rabbits.Salmonella LPS O-antigens are obtained commercially. S. Typhimuriumouter membrane proteins (SOMPs) are purified from strain χ9424 that hasbeen engineered to be unable to produce flagella, all in vitro-expressedpilus antigens, LPS O-antigen and several capsules. Other SalmonellaOMPs are purified from correspondent O-antigen mutants (147).

Statistics:

The SAS program is used to do statistical tests and power analysis toevaluate animal numbers.

Example 3: Construction of Plasmids with Sugar-Regulated Synthesis ofGFP to Enable Determination of Whether a Strain Unable to Metabolize aSugar is Able to Take Up that Sugar to Enable Regulation of a Gene orGene Sequence within that Strain

FIG. 1 diagrams three plasmids pYA3700, pYA5351 and pG8R74 that possessthe araC P_(araBAD), rhaRS-P_(rhaB) and xylR-P_(xylA) cassettes,respectively, as sources of DNA encoding these cassettes to enablegeneration of suicide vectors with fusion of a selected regulatorycassette to a gene of choice in place of the promoter for that gene.These manipulations are described in Example 1 and strains withresulting deletion-insertion mutations with sugar regulated geneexpression are described in the following examples.

The nucleotide sequence of the rhaRS-P_(rhaB) cassette in pYA5351 is asfollows:

(SEQ ID NO: 11) GGGCGAATTCGAGCTCGGTACCCTCGAGGCTGAATTTCATTACGACCAGTCTAAA AAGCGCCTGAATTCGCGACCTTCTCGTTACTGACAGGAAAATGGGCCATTGGCAA CCAGGGAAAGATGAACGTGATGATGTTCACAATTTGCTGAATTGTGGTGATGTGA TGCTCACCGCATTTCCTGAAAATTCACGCTGTATCTTGAAAAATCGACGTTTTTTA CGTGGTTTTCCGTCGAAAATTTAAGGTAAGAACCTGACCTCGTGATTACTATTTC GCCGTGTTGACGACATCAGGAGGCCAGTATGACCGTATTACATAGTGTGGATTTT TTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATT TTCCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGG TATTCATGTGTTTAATGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTC GTACGCGATCATGATCGGCATCTGTATGAACATACCGATAATCTGTGTCTGACCA ATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCAGTT GCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAG CGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGA AAATGATTTACCCTCGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCT TGCTGCGTAAAAGCAGTTTGCAGGAGAACCTGGAAAACAGCGCATCACGTCTCA ACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGAGGTGAATTGGGATGC CGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGCAG CAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCC CGACATCTGCTACGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTG GATTCAGCGACAGTAACCACTTTTCGACGCTTTTTCGCCGAGAGTTTAACTGGTC ACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAACGCGAATCTTCT CAACGTATTTGTACGCCATATTGCGAATAATCAACTTCGTTCTCTGGCCGAGGTA GCCACGGTGGCGCATCAGTTAAAACTTCTCAAAGATGATTTTTTTGCCAGCGACC AGCAGGCAGTCGCTGTGGCTGACCGTTATCCGCAAGATGTCTTTGCTGAACATAC ACATGATTTTTGTGAGCTGGTGATTGTCTGGCGCGGTAATGGCCTGCATGTACTC AACGATCGCCCTTATCGCATTACCCGTGGCGATCTCTTTTACATTCATGCTGACGA TAAACACTCCTACGCTTCCGTTAACGATCTGGTTTTGCAGAATATTATTTATTGCC CGGAGCGTCTGAAGCTGAATCTTGACTGGCAGGGGGCGATTCCGGGATTTAACG CCAGCGCAGGGCAACCACACTGGCGCTTAGGTAGCATGGGGATGGCGCAGGCGC GGCAGGTTATCGGTCAGCTTGAGCATGAAAGTAGTCAGCATGTGCCGTTTGCTAA CGAAATGGCTGAGTTGCTGTTCGGGCAGTTGGTGATGTTGCTGAATCGCCATCGT TACACCAGTGATTCGTTGCCGCCAACATCCAGCGAAACGTTGCTGGATAAGCTGA TTACCCGGCTGGCGGCTAGCCTGAAAAGTCCCTTTGCGCTGGATAAATTTTGTGA TGAGGCATCGTGCAGTGAGCGCGTTTTGCGTCAGCAATTTCGCCAGCAGACTGGA ATGACCATCAATCAATATCTGCGACAGGTCAGAGTGTGTCATGCGCAATATCTTC TCCAGCATAGCCGCCTGTTAATCAGTGATATTTCGACCGAATGTGGCTTTGAAGA TAGTAACTATTTTTCGGTGGTGTTTACCCGGGAAACCGGGATGACGCCCAGCCAG TGGCGTCATCTCAATTCGCAGAAAGATTAATCTAGATAAATAAAAGCAGTTTACA ACTCCTAGAATTGTGAATATATTATCACAATTCTAGGATAGAATAATAAAAGATC TCTGCAGGCATGCAAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTA ATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCT AACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC GTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGC GGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAAC CGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC ATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT GGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCG GGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATT GCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCG GTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCG GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAG CAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATT TCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC TATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACG GTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGC GGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT ATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGC CATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTT CGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGG TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTA ATACGACTCACTATA. 

The nucleotide sequence of the xylR-P_(xylA) cassette in pG8R74 is asfollows:

(SEQ ID NO: 12) GGGCGAATTCGAGCTCGGTACCCTCGAGTCCATAATCAGGTAATGCCGCGGGTG ATGGATGATGTCGTAATATTGGGCACTCCCTTTCAGTTGCTCAATTATGTTATTTC ACACTGCTATTGAGATAATTCACAAGTGTGCGCTCGCTCGCAAAATAAAATGGA ATGATGAAACTGGGTAATTCCGCTAGCttttgataaaaattttctcaaagccggttacgtattaccggttttgagt ttttgcatgattcagcaggaaaagaaccatgtttactaaacgtcaccgcatcacattactgttcaatgccaataaagcctatgaccggcag gtagtagaaggcgtaggggaatatttacaggcgtcacaatcggaatgggatattttcattgaagaagatttccgcgcccgcattgataaa atcaaggactggttaggagatggcgtcattgccgacttcgacgacaaacagatcgagcaagcgctggctgatgtcgacgtecccattg ttggggttggcggctcgtatcaccttgcagaaagttacccacccgttcattacattgccaccgataactatgcgctggttgaaagcgcatt tttgcatttaaaagagaaaggcgttaaccgctttgattttatggtatccggaatcaageggcaaacgttgggccactgagcgcgaatat gcatttcgtcagcttgtcgccgaagaaaagtatcgcggagtggtttatcaggggttagaaaccgcgccagagaactggcaacacgcg caaaatcggctggcagactggctacaaacgctaccaccgcaaaccgggattattgccgttactgacgcccgagcgcggcatattctg caagtatgtgaacatctacatattcccgtaccggaaaaattatgcgtgattggcatcgataacgaagaactgacccgctatctgtcgcgt gtcgccctttcttcggtcgctcagggcgcgcggcaaatgggctatcaggcggcaaaactgttgcatcgattattagataaagaagaaat gccgctacagcgaattttggtcccaccagttcgcgtcattgaacggcgctcaacagattatcgctcgctgaccgatcccgccgttattca ggccatgcattacattcgtaatcacgcctgtaaagggattaaagtggatcaggtactggatgeggtegggatctcgcgctccaatcttga gaagcgttttaaagaagaggtgggtgaaaccatccatgccatgattcatgccgagaagctggagaaagcgcgcagtctgctgatttca accaccttgtcgatcaatgagatatcgcaaatgtgcggttatccatcgctgcaatatttctactctgtttttaaaaaagcatatgacacgacgccaaaagagtatcgcgatgtaaatagcgaggtcatgttgtaatTCTAGAtaaataaaagcagtttacaactcctagaattgtgaatat attatcacaattctaggatagaataataaaagatctctgcagGCATGCAAGCTTGAGTATTCTATAGTGTCA CCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGG TGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGA GAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGC GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTT CGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGC TGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTAT CGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCG GTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAG TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAG CTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAA ATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATC AGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAAC CAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC ATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA GTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCC CCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAG TAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGT CATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACG GGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATG TAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTC TGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC AAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAA CCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCG TCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAG ACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGC GCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAG CAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTA AGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGG GAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGA TGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATA 

FIG. 2 depicts the plasmids pG8R115, pG8R116 and pG8R192 that causesynthesis of GFP to be dependent on the presence of arabinose, rhamnoseor xylose, respectively. These plasmids can be electroporated into anystrain of multiple bacterial species by selection for ampicillinresistance and then screened for synthesis of GFP in the presence of thesugar of interest and the cessation of GFP synthesis in the absence ofthe sugar. If GFP synthesis is observed, then it is possible toconstruct mutant strains in which a promoter for a gene of interest hasbeen deleted and replaced by a araC P_(araBAD), rhaRS-P_(rhaB) orxylR-P_(xylA) cassette so that gene expression is now dependent on thepresence of arabinose or rhamnose or xylose, respectively. It should benoted that this capability is very useful when the bacterial strain orspecies of interest is unable to metabolize or grow on arabinose,rhamnose or xylose such that is unknown whether these sugars can betransported into the bacterial cells that would be necessary is one isto use the presence of that sugar for the expression of genes in thatbacterial strain or species.

Example 4: Isolation and Characterization of Strains with galE Mutationsto Enable Functional Reversible Synthesis of LPS Dependent on Presenceof Free Galactose. Construction of Vaccine Vector Strains with galEMutations to Enable Regulated Synthesis of UDP-Gal Needed for Synthesisof LPS O-Antigen Side Chains and the LPS Outer Core and Enable Growth inPresence of Galactose without Toxicity and Potential Selection ofGalactose-Resistant Variants to Reduce Immunogenicity

The gene galE encodes UDP-galactose 4-epimerase, which interconvertsUDP-galactose and UDP-glucose. As a part of galactose catabolism, galEwas related to both galactose synthesis and degradation. All surfaceliposolysaccharide (LPS) of Salmonella species contain galactose units,in the LPS core and in the LPS O-antigen side chain. Since UDP-galactoseis the precursor of the galactose units in LPS, the galE mutantsynthesizes core-defective or “rough” LPS unless exogenous galactose isprovided (47). The galE mutant of S. Typhimurium is avirulent andconfers protection against virulent S. Typhimurium challenge in mice(47, 50, 155). Some of the galE mutants of S. Choleraesius are alsoavirulent (156, 157). The only licensed live attenuation bacterialvaccine approved for human use is S. Typhi Ty21a with a galE mutation aswell as other mutations (30, 158, 159). S. Typhi Ty21a was onlypartially attenuated and confers moderate protective immunity againsttyphoid in human field trials (30, 47, 160-163).

However, a single galE mutation in S. Typhi still enables virulence forhumans and only provides moderate protection against typhoid (30, 47,161, 163, 164) as also observed with the galE mutants of S. Choleraesuis(165). Besides, Salmonella galE mutants are sensitive to galactose toinduce lysis in vitro (48, 166, 167). Even a defined deletion of galE inS. Typhimurium confers sensitivity to galactose-induced lysis, which isan undesirable attribute to the vaccine. Licensed strain Ty21a has thisunfavored attribute too. galE mutants lack the enzyme UDP galactose4-epimerase but keep the ability to take up galactose from exogenoussources through galactose transporters (168, 169). When grown in thepresence of galactose, the galactokinase and galactose-1-phosphateuridyltransferase, encoded by genes galK and galT, respectively, cansynthesize UDP-galactose from galactose via galactose-1-phosphate andlead to cell growth arrest and even lysis. The exact lytic mechanism isunknown, but death is correlated to the growth and intracellularaccumulation of galactose metabolites, especially the accumulation ofgalactose 1-phosphate and UDP-galactose due to galactokinase activity(30, 48). The accumulation of UDP-D-galactose leads to the growtharrested due to low availability of CTP and UTP, which results inreduced RNA synthesis (49). The galE mutants grow poorly and theirviability are significantly reduced by lyophilization (50). Theavirulence of galE mutants is chiefly due to the incomplete cell walllipopolysaccharide and to galactose-induced bacterial cell lysis (30).It will select for galactose-resistance and Gal⁺ phenotype (170). Thelysis can happen at galactose concentrations as low as 0.002% (166).Galactose-induced lysis occurs in strain Ty21a in vitro at ≥6 mMgalactose (30, 47) whereas Ty2 with a defined galE mutation is even moresensitive to ≥0.06 mM galactose (50, 163). Growth of galE mutants in thepresence of galactose also selects for galactose-resistant strains thatlose the ability to show the reversible rough to smooth variationdependent on supply of galactose (170). Glucose can protectgalactose-sensitive galE strains from lysing by catabolite repression tothe extent that lysis levels of galactose intermediates cannotaccumulate (167, 171). Lowering the galactokinase activity may also givethe strain resistance to galactose (48, 166, 167). Thus, to conquer thisproblem and extend the usage of galE mutations conferring a reversiblerough-smooth variation that serves as a means for regulated delayedattenuation for vaccines, we constructed a strain with a new galEmutation with increased resistance to galactose and yet displaying theregulated attenuation dependent of the presence of added galactose,which is unavailable in vivo. We then evaluated the inclusion of thismutation in S. Typhimurium vaccine strains.

Three strains with different galE mutations were generated. Strain χ4094has a galactose sensitive galE496 mutation, as seen with most galEmutants. Strain χ4700 has an uncharacterized deletion mutationΔ(galE-uvrB)-1005 which enables strains to be insensitive to galactose.Strain χ9792 has a galactose insensitive Δ(galE-ybhC)-851 mutation,which deletes 11 gene sequences from galE to ybhC (FIG. 3A). The strainrequires 0.001% galactose in growth media to form complex LPS O-antigen(FIG. 3B) in either Nutrient broth or LB broth.

To determine whether addition of galactose affects the growth ofSalmonella strains with different galE mutations, growth experimentswere performed. The first experiment evaluated the final ODs ofovernight cultures with varying galactose concentrations in LB broth orNB broth. It should be noted that NB broth is devoid of all sugars suchthat there can be no interference in results due to trace amounts ofgalactose. In LB media, the ODs of the overnight culture of χ4094 is1.088 with 0.001% galactose, but drops to 0.159 with 0.01% galactose.The ODs of χ4700 and χ9792 were not significantly affected by varyingconcentrations of galactose. Similar trends were observed when galEmutants were grown in NB broth with varying galactose concentrations(FIG. 3C). Overall the data confirms χ9792 (Δ(galE-ybhC)-851)) is not assensitive to galactose as χ4094.

A second experiment evaluated growth of the strains during a 7-hourperiod in growth media with varying galactose concentrations (FIGS.4A-4H). An overnight culture of each strain was grown in NB brothwithout galactose. A subculture was made by dilution at 1:100 intoprewarmed 3 ml NB broth with varying percent concentrations of galactose(0, 0.001, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5). The cultures were incubatedat 37° C. with shaking. Optical densities were measured and recordedevery 1 hour. Without galactose, all strains grows similarly. Strainχ4094 has the galactose sensitive galE496 mutation. Strain χ4700 has thegalactose insensitive Δ(galE-uvrB)-1005 mutation. Strain χ9792 has thegalactose insensitive Δ(galE-ybhC)-851 mutation. Strain χ11015 has thegalactose insensitive Δ(galE-ybhC)-851 ΔgalP211 mutation. Strain χ11141has the galactose insensitive Δ(galE-ybhC)-851 ΔgalP211 ΔmglBACmutation. FIGS. 4A-4H showed that mutations ΔgalP211 and ΔmglBAC helpthe strain reach higher ODs.

As shown in FIG. 4B, strain χ4094 grows for 2 hours and then starts tolyse even with 0.001% galactose. With the increasing galactoseconcentrations, the starting time for lysis was reduced. Both strainsχ4700 and χ9792 can tolerate galactose as high as 0.5% withoutcompromising growth (see FIGS. 4A-4H). These results demonstrate thathigh concentrations of galactose do not inhibit the growth andcolonization of a strain with the Δ(galE-ybhC) mutation. The improvedgalactose tolerance enables the strain to display higher tissuecolonization than the strain with the sensitive galE496 mutation at day6 (FIG. 5) following oral inoculation of mice. The data confirms thatthe Δ(galE-ybhC)-851 mutation can be used in vaccine strains to enable areversible rough-smooth phenotype dependent of the presence of galactosein the growth medium and will confer an additional means of regulateddelayed attenuation in vivo since free non-phosphorylated galactose isnot present in animal tissues.

Example 5: Construction and Evaluation of Group B RASV S. TyphimuriumStrains with Rhamnose-Regulated Delayed O-Antigen Synthesis,Mannose-Regulated O-Antigen Side Chain Synthesis and Arabinose-RegulatedProduction of GMMA, or Outer Membrane Vesicles, Synthesizing ProtectiveAntigens In Vivo

O-antigen ligase WaaL is necessary to ligate polysaccharide to the lipidA-core moiety. Mutation of waaL results in an intact core with noO-antigen attached to it (172, 173). We deleted the waaL in the operonand put the rhamnose regulated waaL (ΔP_(rhaBAD) waaL) in the pagL genesince the pagL mutation does not impair Salmonella virulence (174).Rhamnose will replace arabinose to achieve down-regulation of O-antigensynthesis in vivo because a relatively high concentration of rhamnose isnecessary to activate this promoter (175). RASV strains withrhamnose-regulated waaL will synthesis normal LPS in the presence ofrhamnose in vitro, but form rough LPS due to the absence of O-antigenligase in vivo. This strategy exposes the conserved LPS coreoligosaccharide and enhance production of conserved OMPs, includingporins (176, 177), result in more effective presentation of conservedOMPs to the host immune system for enhancing immunogenicity and aid inproduction of a cross-protective immune response against heterologousbacteria (173).

The mutant strain is attenuated to 10⁹ CFU and provides protectionagainst both S. Typhimurium and S. Enteritidis challenge at 10⁹ CFU(Table 1). However, this mutation is not fully attenuated as it stillcauses death at 10⁹. Therefore, a Δpmi mutation is also included asdouble shutoff of O-antigen synthesis in strain χ12339. RASVs withΔP_(rhaBAD) waaL and Δpmi mutations need both rhamnose and mannose toform complete O-antigen. To further increase the protection, a ΔP_(fur)mutation is included to up-regulate IROMPs in vivo to enhance theinduction of cross-protective immunity to enteric pathogens as was donein strain χ12362.

TABLE 1 Oral immunization of BALB/c mice (6-8 weeks) with strain_(χ)12337 (ΔwaaL ΔpagL:: TT rhaSR P_(rhaBAD) waaL) and with survivorschallenged orally with 10⁹ wild-type S. Typhimurium χ3761^(a) and S.Enteritidis χ3550^(b) 30 days later. Immunization data Challenge dataBacterial strain No. of No. of for Dose survivors/total survivors/totalimmunization (CFU) no. of mice no. of mice S. 1.46 × 10⁹ 4/5 4/4^(a)Typhimurium 1.64 × 10⁵ 5/5 4/5^(a) _(χ)12337 1.46-1.64 × 10⁸     10/1010/10^(b) 1.64 × 10⁷ 5/5 3/5^(b) 1.64 × 10⁶ 5/5 3/5^(b) *Strain wasgrown in LB broth with 0.1% rhamnose.

All mutations are dedicated to increase the presentation of conservedproteins to aid in the induction of cross-protective immunity andachieve regulated delayed attenuation. As a tolR mutation can increaseGMMA or outer membrane vesicles production (100, 101), candidate RASVshave been further modified by introduction of an arabinose-regulatedtolR mutation (ΔP_(tolR)::TT araC P_(araBAD) tolR, simplified asΔP_(tolR) thereafter) to further up-regulate GMMA or outer membranevesicles in vivo to maximally induce antibodies cross-reactive to theOMPs of other Salmonella serovars. Furthermore, plasmids encodingprotective antigens will be introduced in the vaccine strains toevaluate protective immunity. Since candidate antigen genes are eitherrepressed or expressed at low levels in vivo (106), overproduction ofthese antigens will facilitate their presentation (106, 108).

The final strain will need arabinose, mannose and rhamnose to behave aswild-type and achieve attenuation in vivo gradually (Table 2). Rhamnose-and mannose-regulated genes will lose their function first, to exposesurface antigens, and then arabinose-regulated genes shut off willincrease GMMA or outer membrane vesicles.

TABLE 2 Phenotypes associated with key mutations in RASV strainsMutation Phenotype ΔwaaL/ΔpagL::TT rhaSR P_(BAD) Deletion of theO-antigen ligase gene waaL, insertion waaL of rhamnose-regulated waaL topagL gene position and deletion of the pagL gene, enable the synthesisof WaaL dependent on the presence of rhamnose in growth medium fornormal LPS as wild type in vitro and ceases to be synthesized in vivodue to the absence of rhamnose, resulting in incomplete O- antigensynthesis and attenuation. Δpmi Deletion of phosphomannose isomerasegene to convert fructose to mannose necessary for synthesis of LPSO-antigen side chains. LPS O-antigen can be synthesized during in vitrogrowth by exogenous mannose in the growth medium for exhibiting nearlywild-type attributes for survival and colonization of lymphoid tissuesat the time of immunization and lost after five to ten cell divisions invivo and become avirulent due to inability to synthesize the LPS O-antigen side chains for the absence of free non- phosphorylated mannoseand also become sensitive to complement-mediated cytotoxicity andsusceptible to phagocytosis by macrophages. ΔP_(fur)::TT araC P_(BAD)fur The fur gene encodes a repressor that represses all genes involvedin iron acquisition in presence of free iron. When iron concentrationsbecome low, as in animal host tissues beyond the intestinal wallbarrier, the Fur ceases to be synthesized and constitutive synthesis ofIROMPs commences. This mutation enables turn on the fur gene witharabinose in vitro and turn off in the absence of arabinose in vivo foroverexpression of IROMPs in vivo and leads to attenuation. ΔP_(tolR)::TTaraC P_(BAD) tolR The deletion-insertion mutation eliminates TolRsynthesis and up-regulate GMMA or outer membrane vesicles production invivo due to the absence of arabinose. ΔrelA::araC P_(BAD) lacI TT Thedeletion-insertion mutation eliminates RelA which governs synthesis ofppGpp and couples growth to protein synthesis. The araC P_(BAD) lacIinsertion causes an arabinose-dependent synthesis of the LacI repressorin vitro, which governs the express of genes encoding protective proteinantigens encoded on plasmids, and enable antigen production in vivo dueto depletion of LacI in vivo. ΔasdA For balanced-lethal system andmaintains complete sensitivity of RASV to all antibiotics. Δ(wza-wcaM)Eliminates twenty enzymes needed to synthesize severalexopolysaccharides that promote biofilm formation and synthesis ofGDP-fucose required for colanic acid synthesis (178), which protectscells undergoing cell wall-less death from lysing (179) Δ(galE-ybhC)Deletion of UDP-glucose 4-epimerase gene to interconvert UDP-galactoseand UDP-glucose necessary for synthesis of LPS O-antigen core. LPSO-antigen can be synthesized during in vitro growth by exogenousgalactose in the growth medium for exhibiting nearly wild-typeattributes for survival and colonization of lymphoid tissues at the timeof immunization and lost after several cell divisions in vivo and becomeavirulent due to inability to synthesize the LPS O-antigen core due tothe absence of free non-phosphorylated galactose and also becomesensitive to complement-mediated cytotoxicity and susceptible tophagocytosis by macrophages. Mutants with this mutation can toleratehigh concentration of galactose to 0.5% Note: Δ = Deletion of geneticsequence; P = Promoter for RNA polymerase recognition and binding; :: =insertion of DNA or gene sequence; TT = Transcription terminator.

Strain Construction.

Strain χ12470 is generated by using Strain χ12337 and subsequentlyadding mutations Δpmi, ΔP_(fur) and ΔP_(tolR) sequentially (Table 2).Mutations ΔrelA::araC P_(araBAD) lacI TT (ΔrelA) for RDPS (180), ΔasdAfor the balanced-lethal system, and Δ(wza-wcaM) to eliminate synthesisof exopolysaccharides (Table 2) are introduced result in strain χ12465to facilitate its use as a vector. A ΔP_(tolR) mutation—is added togenerate strain χ12473. After confirmation of final strain by phenotypicand PCR analysis, Asd⁺ plasmids carrying an individual antigen gene areintroduced into the strain. The corresponding antigen gene will bedeleted using a suicide vector (144, 181) from the chromosome to preventpotential recombination between genes on the chromosome and plasmid.Membrane integrity, OMVs production (101), presence and stability of allphenotypic traits of strains are thoroughly investigated. The sugarregulated promoters or SD or start codon may be switched to regulate theproduction of O-antigen ligase, Fur, TolR in vitro to balance theimmunogenicity and attenuation (75).

Plasmid Construction.

Since all the antigens are surface exposed or secreted, natural genesequences are expressed using the balanced-lethal Asd⁺ vector pYA3342(P_(trc), pBR ori) (147). The presence of RDPS will repress the antigengene expression in vitro by arabinose, but up-regulate in vivo (180). Ashift to a low copy plasmid pYA3337 (P_(trc), pSC101 ori) (182) orpYA3332 (P_(trc), p15A ori) (183) is performed if overproduction leadsto a metabolic burden as indicted by significantly slower growth.

All the genes exceptfliC are used according to their natural sequence. Atruncated FliC180, which deletes the 180 amino acids encoding theantigenically variable serovar-specific hypervariable domain of theflagellin antigen, is used to reduce the induction of antibody titers toserovar-specific antigens and increase the cross protection againstconserved domain of flagellin. The FliC180 protein retains the conservedN- and C-terminal regions that interact with TLR5 to recruit/stimulateinnate immune responses (184, 185) and the CD4-dependent T-cell epitopes(186). The individual antigen is tested first, followed by testing ofmultiple antigens using plasmid encoding several antigens as an operon(183, 187, 188) or with multiple genes that are independently regulated(189-193). The recF mutation will be incorporated to reducerecombination between antigens on plasmid (194).

In Vitro Evaluation of RASVs Expressing Protective Antigen Genes.

The ability of the RASV strains to synthesize and secrete protectiveantigen is analyzed by conducting cell fractionation studies todetermine the amount of antigen present in the cytoplasm, periplasm andsupernatant fractions by western blot. Strains are grown in Luria Broth(LB) to an OD₆₀₀ of 0.8 at 37° C. and centrifuged. The supernatant fluidis saved for analysis of secreted proteins. Periplasmic and cytoplasmicfractions are prepared by a lysozyme-osmotic shock method (147, 195,196). Equal volumes of periplasmic, cytoplasmic and supernatantfractions and total lysate samples are analyzed via western blots probedwith correspondent antibody. Tissue culture experiments are performed toevaluate antigen translocation into mouse macrophage-like cell lines,J774.A and/or P388D1 by western blots and immunofluorescence (197-199).

Animal Experiments.

BALB/c female mice, six to eight weeks of age, are used and housed inBSL2 containment with filter bonnet covered cages. Typical experimentsinclude groups of fifteen mice for challenge (repeat once) (200).Additional mice are used to determine colonization and for harvestingspleens for immunological analyses. Colonization and immunogenicity isevaluated for all constructions synthesizing Salmonella conservedprotective antigens.

Mice are immunized orally on day 0 with a dose of ˜10⁹ CFU RASVs,boosted with the same dose 1 week after, and orally challenged at week 4with 100×LD₅₀ virulent Salmonella strain according to standardprocedures (200). LD₅₀s of wild type strains are known or are evaluated.Morbidity and mortality are recorded daily. First, strains carrying eachindividual antigen with PBS control against S. enteritidis challenge arecompared. If protection is observed in this test, subsequent studies aredone to determine the cross protection against other Salmonellaserovars. Blood, PP, liver and spleen are harvested from challenged micefor Salmonella enumeration in tissues to determine the kinetics ofelimination of viable Salmonella as a function of time after challengeand monitor post-challenge immune responses.

Measurement of Immune Responses Conferred by RASVs SynthesizingProtective Antigens.

Serum IgG and mucosal SIgA responses from vaginal washes in immunizedmice are evaluated by ELISA using the protective antigens, OMPs, IROMPsand LPS from different serovars at 2 and 4 weeks, as well as IgG1 andIgG2a titers to distinguish between Th1 and Th2 responses. At 4 weekspost-immunization, the splenocyte responses to stimulation with purifiedSalmonella antigens or Salmonella are determined for measurement ofT-cell immunity by ELISPOT to determine the CD4 T-cell profile thatproduce IL-4, IFN-γ and IL-17 (201). Since the amount of secreted IgAobtained in vaginal washes may not accurately reflect the mucosalresponse in the gut, the number of IgA secreting cells present in thelamina propria of the intestine is measured by antigen-specific IgAELISPOT. Sera are collected for cytokine assays using a multiplex assayat 24, 48 and 72 h post-challenge using the Bio-plex Protein ArraySystem (BIO-RAD) according to the manufacturer's instructions (202). Thecytokines IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF-α, IL-21 and IL-23are measured as a result of co-cultures of the T lymphocytes withSalmonella antigens to determine the T-cell differentiation pathwaysamong Th1/Th2/Th17/Tfh using a Bioplex assay (202-204). Specially, IL-1βand IL-18 are monitored for bacterial multiplication in the liver andspleen (41, 205, 206), TNF-α and IL-6 for LPS induced cytokines (207,208). Flow cytometry is used to determine distribution of the memory Band T cells in mouse PBMCs and tissues (209, 210) and T-cellproliferation by CFSE staining (211-218).

Example 6: Construction and Evaluation of Group D RASV S. EnteritidisStrains with Rhamnose-Regulated Delayed O-Antigen Synthesis,Mannose-Regulated O-Antigen Side Chain Synthesis and Arabinose-RegulatedProduction of GMMA or Outer Membrane Vesicles Synthesizing ProtectiveAntigens In Vivo

RASV-Enteritidis strains with the same features as RASV-Typhimuriumsynthesizing protective antigens will be constructed and evaluated inparallel with RASV-Typhimurium as a complementary strategy. The suicidevectors used for S. Typhimurium may be used for S. Enteritidis due tothe high homology between the two serovars. Since there is no animalmodel for S. Typhi, this work also facilitates the translation ofresults to S. typhi because both of them are group D Salmonella. It mayalso help to use as a bivalent vaccine or for prime-boost immunizationagainst the majority of NTS infections (219).

Construction of RASV Enteritidis Vaccine Strains

Similar strategies are used to construct S. enteritidis strains with themutations, ΔwaaL, ΔP_(rhaBAD) waaL, Δpmi, ΔP_(fur) and ΔP_(tolR),derived from S. enteritidis χ3550, to generate vaccine strain B 1. AΔP_(tolR) mutation will be added to strain χ12457, derived from strainχ3550 with mutations ΔwaaL, ΔP_(rhaBA) waaL, Δpmi and ΔP_(fur), togenerate vaccine strain B. The virulence of the resulting strain isassessed in BALB/c mice. Mutations to reduce the lipid A toxicity areintroduced if the strain is still virulent (220, 221). Providing thestrain is attenuated as expected, immunized mice are challenged orallywith 100×LD₅₀ of wild-type S. Typhimurium strain χ3761. If protection isobserved, subsequent studies determine the cross protection againstother Salmonella serovars. Assuming the strain is adequately attenuatedand provides some protection, ΔrelA and ΔasdA are introduced to generatestrain B2 to facilitate its use as a vector.

In Vitro Evaluation of RASV Enteritidis Antigen Delivery Vector.

The best vector from Example 3 is introduced into strain B2. Theresulting recombinant strain is evaluated for antigen synthesis, plasmidstability and other characters in vitro and in vivo essentially asdescribed in Example 3.

Animal Experiments and Measurement of Immune Responses Conferred byRASV-Enteritidis Synthesizing Protective Antigens.

Similar procedures and tests are carried out as in Example 3 except S.Typhimurium will be challenged first, and then other serovars.

Example 7. Improved Performance of RASV Against Clostridiumperfringens-Induced Necrotic Enteritis in Broiler Chickens with StrainsDisplaying the Regulated Delayed Lysis In Vivo Phenotype and OtherAttenuation and Protective Antigen Synthesis Attributes Dependent on TwoVersus Three Sugar Regulated Properties

To determine the protective effects of a recombinant bacterial strain orRASV comprising three sugar-regulatable attribute systems versus twosugar-regulatable attribute systems, the following experiments wereperformed.

I. Comparative Immunogenicity and Protective Immunity of 11802 Versus12341

Broiler chickens were orally immunized with one of the followingSalmonella enterica strains:

-   -   χ11802 ΔP_(murA25)::TT araC P_(BAD) murA ΔasdA27::TT araC        P_(BAD) c2 Δpmi-2426 Δ(wza-wcaM)-8 ΔrelA198::araC P_(BAD) lacI        TT ΔrecF126 (arabinose- and mannose-regulatable phenotypes)        comprising pYA5112 (described in Jiang et al. (2015) Avian        Diseases 59:475-85 (188), the entire contents of which are        incorporated herein by reference) encoding an operon for        synthesis of PlcC and a NetB fusion as C. perfringens protective        antigens.    -   χ12341 ΔP_(murA25)::TT araC P_(BAD) murA ΔasdA27::TT araC        P_(BAD) c2 Δpmi-2426 ΔwaaL46 ΔpagL64::TT rhaRS P_(rhaBAD) waaL        Δ(wza-wcaM)-8 ΔrelA197::araC P_(BAD) lacI TT ΔrecF126 ΔsifA26        (arabinose-, mannose- and rhamnose-regulatable phenotypes)        comprising pYA3681 as the empty vector control or pYA5112        encoding an operon for synthesis of PlcC and a NetB fusion as C.        perfringens protective antigens.

The study consisted of 48 cages starting with 384 chicks. The treatmentswere replicated in 6 blocks of 8 cages each. The study began when thebirds were placed (day of hatch) (DOT 0) at which time they wereallocated to the experimental cages. No birds were replaced during thecourse of the study.

TABLE 3 Treatment Groups Coccidial Clostridium Treatment Challengeperfringens Cages/Trt T1 Nonmedicated DOT 14 No 8 T2 Nonmedicated DOT 14DOT 19, 20, and 21 8 T3 Vaccine 1* DOT 14 DOT 19, 20, and 21 8 T4Vaccine 2* DOT 14 DOT 19, 20, and 21 8 T5 χ12341 DOT 14 DOT 19, 20, and21 8 comprising pYA3681 (Vector Control)* T6 BMD 50 g/t DOT 14 DOT 19,20, and 21 8 *Oral gavage on DOT 0. Vaccine 1: χ11802(pYA5112); Vaccine2: χ12341(pYA5112)

Experimental Ration

An unmedicated chicken starter compounded with feedstuffs commonly usedin the United States was formulated. The diet was representative of alocal commercial formulation and calculated analyses met or exceeded NRCbroiler starter requirements. The diet formulation was included in thesource data. Experimental treatment feeds were prepared from this basalstarter feed. Quantities of all basal feed and test articles used toprepare treatment batches were documented. Treatment feeds were mixed toassure a uniform distribution of respective test article. The mixer wasflushed to prevent cross contamination. The feed was transferred toBuilding #2 and distributed among cages of the same treatment. Atplacement, the birds were fed the treatment feeds. This ration (in mashform) was fed during the study. Feed was weighed in on DOT 0 andremaining feed was weighed on DOT 14, 21, and 28.

Feed Samples

One each from the beginning, middle, and end of each batch of treatmentdiet was collected and mixed to form a composite sample. One sample wastaken from the composite for each treatment and retained for a period ofsix (6) months after study completion for potential feed analysis.

Animals

Day of hatch male broiler chicks were obtained from Cobb-Vantress,Cleveland, Ga. The strain was Cobb 500. Breeder flock information wasrecorded. At the hatchery, the birds were sexed and received routinevaccinations. Only healthy appearing chicks were used in the study. Eachcage started with 8 chicks (DOT 0). All birds were weighed on DOT 0, 14,21, and 28.

Strain Administration

Bacterial strains were administered at DOT 0 to each chick in TreatmentGroups 3, 4, and 5 via oral gavaged with ˜5×108 CFU/chick in a volume of0.1 mL.

Disease Induction

On DOT 14, all birds were orally inoculated with ˜5,000 oocysts of E.maxima.

Starting on DOT 19 all birds (except Treatment 1) were given a brothculture of C. perfringens ˜10⁸ CFU/ml. There were no feed removed inthis study. The birds were administered 0.1 ml by oral gavage of a freshbroth culture once daily for 3 days (on DOTs 19, 20, and 21).

Clostridium perfringens Challenge Growth

The challenge strain used was Clostridium perfringens #6 (Hofacre, etal., 1998) (222). It was inoculated into one (1) liter of thioglycolatebroth supplemented with 5% beef extract and incubated at 37° C. for 15hours.

Necrotic Enteritis Intestinal Lesion Scoring

Necrotic enteritis intestinal lesion scoring was performed as describedin Hofacre, et al., 1998 (222). On DOT 21, three birds from each cagefour (4) hours post third Clostridium perfringens challenge wereselected, sacrificed, weighed, and examined for the degree of presenceof Necrotic Enteritis lesions. The scoring was based on a 0 to 3 score,with 0 being normal and 3 being the most severe.

Data Analysis

Statistical analysis of cage weight gain, feed consumption, feedconversion, lesion scores, and NE mortality were calculated. The resultsof the experiment are shown below at Table 4.

TABLE 4 Determining the best vaccine strain genotype Feed Weight GainFeed Weight Gain Conversion (kg) Conversion (kg) NE % NE TreatmentsD0-21 D14-21 D0-21 D14-21 D0-28 D14-28 D0-28 D14-28 Lesions Mortality 1.No Additive, No CP 2.054b 1.626c 0.286a 0.164a 1.958b 1.706b 0.657a0.535a 0.0d 0.0a 2. No Additive, CP 2.585a 2.053ab 0.226b 0.125b 2.241a1.875ab 0.506b 0.405b 0.9a 6.3a 3. χ11802(pYA5112), CP 2.340ab 2.093a0.270ab 0.142ab 2.106ab 1.888a 0.627ab 0.499ab 0.8a 6.3a 4.χ12341(pYA5112), CP 2.161b 1.826bc 0.294a 0.154a 1.937b 1.698b 0.709a0.570a 0.3cd 0.0a 5. Vector Control, CP 2.316ab 2.043ab 0.276ab 0.153a2.060ab 1.854ab 0.649a 0.527a 0.6ab 4.7a 6. BMD 50 g/t, CP 2.235b1.889ab 0.268ab 0.152a 1.981b 1.742ab 0.642a 0.526a 0.5bc 1.6a a: b: c:d:

TABLE 5 Determining the effect of varying doses of RASV χ12341(pYA5112)Feed Weight Gain Feed Weight Gain Conversion (kg) Conversion (kg) NE %NE Treatments D0-21 D14-21 D0-21 D14-21 D0-28 D14-28 D0-28 D14-28Lesions Mortality 1. No Additive, No CP 1.714c 1.842d 0.509a 0.223a1.807b 1.917c 0.710a 0.423a 0.1b  0.0c 2. No Additive, CP 2.329a 3.188a0.354c 0.119e 2.210a 2.517a 0.501c 0.266d 0.4ab 15.6a 3.χ12341(pYA5112), 2.264ab 2.197cd 0.399b 0.179b 2.164a 2.066bc 0.602b0.382ab 0.4a  1.6bc Original titer, CP 4. χ12341(pYA5112), 2.240ab2.444bc 0.390bc 0.149cd 2.121a 2.172bc 0.570bc 0.329bcd 0.5a  1.6bcIntermediate titer, CP 5. χ12341(pYA5112), 2.386a 2.415bc 0.375bc0.159bcd 2.231a 2.134bc 0.571bc 0.355abc 0.5a  6.3b low titer, CP 6.Vector Control, CP 2.307a 2.754b 0.378bc 0.137de 2.165a 2.286ab 0.537bc0.296cd 0.5a  4.7bc 7. BMD 50 g/t, CP 2.040b 2.142cd 0.407b 0.166bc2.039a 2.098bc 0.591bc 0.351abc 0.5a  1.6bc

As shown in Table 4, χ12341(pYA5112) was superior to χ11802(pYA5112)(and the vector and unimmunized controls) in feed conversion efficiencyand weight gain and with lower lesion scores and mortality.

II. Effect of Dose of RASV χ12341(pYA5112).

To assess the effect of dosing of RASV χ12341(pYA5112), the followingexperiment was performed using either low titer (5×10⁷ CFU);intermediate titer (1.5×10⁸ CFU) or the original titer (as describedabove; 5×10⁸ CFU) of the RASV χ12341(pYA5112) bacterial strain.

Materials and Methods

A. Experimental Ration

An unmedicated chicken starter compounded with feedstuffs commonly usedin the United States was formulated. The diet was representative of alocal commercial formulation and calculated analyses met or exceeded NRCbroiler starter requirements. Experimental treatment feeds were preparedfrom this basal starter feed. Quantities of all basal feed and testarticles used to prepare treatment batches were documented. Treatmentfeeds were mixed to assure a uniform distribution of respective testarticle. The mixer was flushed to prevent cross contamination. The feedwas distributed among cages of the same treatment. This ration (in mashform) was fed during the study.

B. Animals

Day of hatch male broiler chicks were obtained from Cobb-Vantress,Cleveland, Ga. The strain was Cobb 500. Breeder flock information wasrecorded. At the hatchery, the birds were sexed and received routinevaccinations. Only healthy appearing chicks were used in the study.Disposition of all birds not used for allocation were documented. Papersor swabs from bottom of all chick boxes were cultured for presence ofSalmonella.

Procedures

a. Bird Allocation and Cage Randomization

The study began when the birds were placed (day of hatch) (DOT 0) atwhich time they were allocated to the experimental cages. No birds werereplaced during the course of the study.

b. Vaccine Administration

Bacterial strains were administered at DOT 0 to each chick in TreatmentGroups 3, 4, 5 and 6 via oral gavaged with ˜5×10⁸ CFU/chick in a volumeof 0.1 mL.

c. Cage Weights

All birds were weighed on DOT 0, 14, 21, and 28. Feed was weighed in onDOT 0 and remaining feed was weighed on DOT 14, 21, and 28.

d. Disease Induction

On DOT 14, all birds were orally inoculated with ˜5,000 oocysts of E.maxima. Starting on DOT 19 all birds (except Treatment 1) were given abroth culture of C. perfringens ˜10⁸ CFU/ml. No feed was removed in thisstudy. The birds were administered 0.1 ml by oral gavage of a freshbroth culture once daily for 3 days (on DOTs 19, 20, and 21).

e. Clostridium perfringens Challenge Growth

The challenge strain used was Clostridium perfringens #6 (as describedin Hofacre, et al., 1998 (222)). It was inoculated into one (1) liter ofthioglycolate broth supplemented with 5% beef extract and incubated at37° C. for 15 hours.

f. Necrotic Enteritis Intestinal Lesion Scoring

On DOT 21, three birds from each cage four (4) hours post thirdClostridium perfringens challenge were selected, sacrificed, weighed,and examined for the degree of presence of Necrotic Enteritis lesions.The scoring was based on a 0 to 3 score, with 0 being normal and 3 beingthe most severe. All of the three lesion score birds were bled for serumstorage.

g. Data Analysis and Results

Statistical analysis of cage weight gain, feed consumption, feedconversion, lesion scores, and mortality were calculated. The results ofthe experiment are shown below at Table 5. As shown in Table 5, theoriginal and intermediate RASV doses were superior to the low dose orthe controls.

III. Effect of Route of Immunization.

To determine the effect of the route of administration of RASVχ12341(pYA5112), chicks were immunized with either a high dose (5×10⁸CFU) or mid dose (1×10⁸ CFU) of the χ12341(pYA5112) bacterial straineither by oral gavage, spray, or orally (in drinking water). Briefly, atDOT 0, each chick was orally gavaged with 0.1 ml of the bacterialstrain, ˜5×10⁸ CFU/chick; On DOT 14, all birds were orally inoculatedwith ˜5,000 oocysts of E. maxima. Starting on DOT 19 all birds (exceptTreatment 1) were given a broth culture of C. perfringens (CP) ˜10⁸CFU/ml once daily for 3 days (on DOTs 19, 20, and 21). On DOT 21, threebirds from each cage four (4) hours post third Clostridium perfringenschallenge were examined for the degree of presence of Necrotic Enteritislesions. The scoring was based on a 0 to 3 score, with 0 being normaland 3 being the most severe.

As shown in Table 6, all routes of immunization were superior to thecontrol unvaccinated group. Moreover, the spray immunization groupresulted in satisfactory performance as compared to the oral gavagegroups. This is commercially important since spray immunization inhatcheries is the preferred and most economical means of immunizationfor poultry.

TABLE 6 Effects of the route of immunization of χ12341(pYA5112) FeedWeight Gain Feed Weight Gain Conversion (kg) Conversion (kg) NE % NETreatments D0-21 D14-21 D0-21 D14-21 D0-28 D14-28 D0-28 D14-28 LesionsMortality 1. No Additive, No 1.683d 1.883d 0.524a 0.240a 1.765c 1.937d0.836a 0.552a 0.0c  0.0c CP 2. No Additive, CP 1.984a 2.729a 0.445bc0.164d 2.212a 3.271a 0.635c 0.353c 0.9a 40.6a 3. χ12341(pYA5112)-1.958ab 2.540ab 0.436c 0.172cd 1.975bc 2.361bc 0.664bc 0.400bc 0.7ab20.3b High dose gavaged, CP 4. χ12341(pYA5112)- 1.814bcd 2.213c 0.468bc0.191bc 1.835bc 2.115bcd 0.770ab 0.496ab 0.4bc 12.5bc Mid dose gavaged,CP 5. χ12341(pYA5112)- 1.905abc 2.493b 0.451bc 0.173bcd 1.855bc 2.189bcd0.773ab 0.478ab 0.9a 20.3b High dose sprayed, CP 6. χ12341(pYA5112)-1.904abc 2.574ab 0.467bc 0.169cd 1.978b 2.508b 0.789a 0.491ab 0.8ab20.3b High dose in drinking H₂O, CP 7. BMD 50 g/t, CP 1.760cd 2.114c0.487ab 0.197b 1.805bc 2.071cd 0.832a 0.542a 0.4bc 17.2b

Example 8. Inability of High Concentrations of One Sugar to Interferewith Ability of Two Other Sugars at Low Concentrations to Regulate GenesNeeded for Survival of RASV Strains with the Regulated Delayed Lysis InVivo Phenotype

χ12341(pYA4763) has an obligate requirement for arabinose to survivesince it has araC P_(araAD)-regulated murA genes both in the chromosomeand in the pYA4763 plasmid. Since the product of the murA gene-encodedenzyme is phosphorylated and since Salmonella cannot take upphosphorylated sugars, the χ12341(pYA4763) strain constitutes anarabinose-dependent lethal construction. Thus any exogenous sugar thatwould block the ability of arabinose to be either taken up byχ12341(pYA4763) or to cause transcription of the murA gene by activationof the P_(araBAD) promoter would result in lethality of χ12341(pYA4763)cells. To determine whether addition of high concentrations (i.e., 1.0%)of any of the three sugars used by χ12341(pYA4763) interferes with theactivities of the other two sugars at lower concentrations (i.e., 0.1%or lower) to enable survival or display of the phenotype regulated, thefollowing experiments were performed. Growth experiments were performedusing buffered Purple broth (to avoid any pH change due sugarutilization).

Briefly, the growth of Salmonella strains χ12341(pYA4763) and χ3761 wasassessed during a 24 h period in growth media with varying sugarconcentrations. Briefly, an overnight culture of each strain was grownin buffered purple broth+0.05% arabinose+0.1% rhamnose+0.1% mannose. Asubculture was made by diluting at 1:100 (FIGS. 6A, 6B, 6G, 6H, 6M, and6N), 1:1,000 (FIGS. 6C, 6D, 6I, 6J, 6O, and 16P), or 1:10,000 (FIGS. 6E,6F, 6K, 6L, 6Q, and 6R) into pre-warmed buffered purple broth withvarying concentrations of arabinose, rhamnose, and mannose. 200 μL ofeach culture was added to an individual well in a 100-well plate induplicate for each strain and sugar condition. The plate was insertedinto the Bioscreen C Automated Microbiology Growth Curve Analysis Systemset at 37° C. and was left to incubate, with shaking, for 24 h. Opticaldensities were measured every 30 min. and compared to a blank to confirmpurity. The figures present the data with all conditions (FIGS. 6A-6F),comparing the conditions that had 1% of one of the three sugars (FIGS.6G-6L) and comparing various concentrations of arabinose (FIGS. 6M-6R).

As shown in FIGS. 6A-6R, the χ12341(pYA4763) bacterial strain grows aswell as wild-type S. Typhimurium UK-1 strain independent of the presenceof any one sugar at a 1.0% concentration and the other two sugars at0.1% or lower concentrations. It should be noted that Purple broth isdevoid of all sugars such that there can be no interference in resultsdue to trace amounts of arabinose, mannose or rhamnose. These resultsdemonstrate that high concentrations of rhamnose or mannose do notinhibit the ability of low concentrations of arabinose to causeexpression of the murA gene since no cell death was observed.

Western blot analysis can be performed to analyze the expression ofgenes encoding products regulated by one of the sugar-regulatablepromoters in the χ12341(pYA4763) strain.

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1. A recombinant derivative of a pathogenic bacterium comprising: a. afirst gene regulated by a first sugar which confers a first phenotype;b. a second gene regulated by a second sugar which confers a secondphenotype; and c. a third gene regulated by a third sugar which confersa third phenotype; wherein the first, second and third phenotypes areselected from the group consisting of:
 1. a regulated-delayedattenuation;
 2. a regulated-delayed expression of an antigen ofinterest;
 3. a regulated-delayed lysis in vivo;
 4. a regulated synthesisof O-antigen;
 5. a regulated synthesis of an O-antigen side chain;
 6. aregulated production of Generalized Modules for Membrane Antigens(GMMA);
 7. regulated enhanced survival to a host stress condition; and8. a regulated production of outer membrane vesicles (OMVs).
 2. Thebacterium of claim 1, wherein the first sugar, second sugar, and thirdsugar are each a different sugar, optionally wherein any one of thefirst, second or third sugar does not interfere with the regulation of agene regulated by a different sugar.
 3. (canceled)
 4. The bacterium ofclaim 1, wherein the first, second, or third sugar is selected from thegroup consisting of arabinose, mannose, xylose, galactose, rhamnose,lactose, and maltose. 5.-6. (canceled)
 7. The bacterium of claim 4,wherein the first, second or third gene is operably-linked to a first,second, or third sugar-regulatable promoter, respectively. 8.-9.(canceled)
 10. The bacterium of claim 1, wherein a gene is modified toenable a reversible synthesis of a sugar-containing molecule thatconfers a sugar regulatable phenotype. optionally wherein the modifiedgene is pmi or galE. 11.-14. (canceled)
 15. The bacterium of claim 1,wherein the phenotype is regulated delayed attenuation, and the geneconferring the phenotype is fur; wherein the phenotype isregulated-delayed expression of an antigen of interest, and the geneconferring the phenotype encodes an antigen of interest; wherein thephenotype is regulated-delayed lysis in vivo, wherein the lysis isenabled to occur in a cytosol due to mutation in a sifA gene, andoptionally wherein the sifA gene is operably linked to anarabinose-regulatable promoter; wherein the phenotype is regulatedsynthesis of O-antigen, and the gene conferring the phenotype isselected from the group consisting of waaG, rfaH, waaJ, wbaP, wzy, waaP,waaO, waaF, waaP, waaC, waaA, waaL and wbaP; wherein the phenotype isproduction of Generalized Modules for Membrane Antigens (GMMA) or outermembrane vesicles, and the gene conferring the phenotype is selectedfrom the group consisting of vbgC, tolQ, tolA, tolR, tolB, paI, andybgF; wherein the phenotype is regulated synthesis of O-antigen sidechain, and the gene conferring the phenotype is tolR; or wherein thefirst phenotype is regulated O-antigen synthesis and the secondphenotype is production of GMMA or outer membrane vesicles. 16.-21.(canceled)
 22. The bacterium of claim 1, wherein the bacterium furthercomprises a gene encoding an antigen of interest not operably-linked toa sugar regulatable promoter.
 23. The bacterium of claim 1, wherein thebacterium comprises a deletion of an endogenous O-antigen synthesisgene, a deletion in an endogenous phosphomannose isomerase gene, adeletion in an endogenous tol-paI system gene, a deletion in a geneencoding an aspartate-semialdehyde dehydrogenase, a deletion in a pagLgene, a deletion in a sifA gene, a deletion in a recF gene, and/or adeletion in a recJ gene. 24.-37. (canceled)
 38. The bacterium of claim4, wherein the first, second or third sugar-regulatable promoter is arhamnose-regulatable promoter, optionally wherein therhamnose-regulatable promoter is rhaSR P_(rhaBAD); or anarabinose-regulatable promoter, wherein the arabinose regulatablepromoter is araC P_(araBAD). 39.-43. (canceled)
 44. The bacterium ofclaim 1, further comprising a nucleic acid encoding a LacI repressor,wherein the LacI repressor is encoded by a lacI gene. 45.-51. (canceled)52. The bacterium of claim 1, further comprising a deletion in a geneencoding an aspartate semialdehyde dehydrogenase, wherein the geneencoding an aspartate semialdehyde dehydrogenase comprises an asd or anasdA gene. 53.-55. (canceled)
 56. The bacterium of claim 1, wherein thegene encoding the antigen of interest is located in a plasmid in thebacterium, and wherein the plasmid further comprises a nucleic acidencoding an aspartate-semialdehyde dehydrogenase. 57.-65. (canceled) 66.The bacterium of claim 1, wherein the bacterium further comprises anantigen of interest operably-linked to a repressor-regulatable promoter.67.-69. (canceled)
 70. The bacterium of claim 66, wherein the antigen ofinterest is an antigen derived from an infectious agent or an antigenassociated with cancer. 71.-76. (canceled)
 77. The bacterium of claim70, wherein said antigen is a C. perfringens antigen, and wherein theantigen comprises NetB, PlcC, antigenic fragments thereof, fusionproteins comprising said antigens, or fusion proteins comprisingantigenic fragments of antigens. 78.-83. (canceled)
 84. The bacterium ofclaim 1, further comprising a deletion in a sifA gene. 85.-95.(canceled)
 96. The bacterium of claim 1, wherein the bacterium is aSalmonella enterica subsp. enterica serovar Paratyphi A bacterium, aSalmonella enterica subsp. enterica serovar Enteritidis bacterium, aSalmonella enterica subsp. enterica serovar Typhi bacterium, aSalmonella enterica subsp. enterica serovar Typhimurium bacterium,Salmonella enterica subsp. enterica serovar Dublin, or Salmonellaenterica subsp. enterica serovar Choleraesuis.
 97. A pharmaceuticalcomposition comprising the recombinant bacterium of claim 1, and apharmaceutically acceptable carrier.
 98. A method for eliciting animmune response against an antigen of interest in a subject, the methodcomprising administering to the subject an effective amount of apharmaceutical composition of claim
 97. 99. A recombinant derivative ofa pathogenic bacterium, wherein the bacterium comprises an araCP_(araBAD)-regulated murA gene; a deletion-insertion mutation thatinactivates the expression of asdA gene and inserts a c2 gene; adeletion in a pmi gene; a deletion in a pagL gene; a rhaRSP_(rhaBAD)-regulated waaL gene; a deletion in a wza-wcaM gene; adeletion-insertion mutation that inactivates the expression of a RelAgene and inserts a lacI gene; a deletion in a recF gene; and a deletionin a sifA gene.